AU2013205033B2 - Novel Methods of Constructing Libraries Comprising Displayed and/or Expressed Members of a Diverse Family of Peptides, Polypeptides or Proteins and the Novel Libraries - Google Patents

Abstract

Methods useful in constructing libraries that collectively display and/or express members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens. z <co w I. <a CO Io uq~ ________r-ill CL' CL I- \o Ile L= -j D 0 CD0 co LU

Description

NOVEL METHODS OF CONSTRUCTING LIBRARIES COMPRISING DISPLAYED AND/OR EXPRESSED MEMBERS OF A DIVERSE FAMILY OF PEPTIDES, POLYPEPTIDES OR PROTEINS AND THE

NOVEL LIBRARIES

RELATED APPLICATION AND TECHNICAL FIELD

This application is a divisional of Australian Patent Application No. 2011253898, which is a divisional of Australian Patent Application No. 2009200092. All of the above referenced applications are hereby incorporated by reference in their entireties .

The present invention relates to libraries of genetic packages that display and/or express a member of a diverse family of peptides, polypeptides or proteins and collectively display and/or express at least a portion of the diversity of the family. In an alternative embodiment, the invention relates to libraries that include a member of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family. In a preferred embodiment, the displayed and/or expressed polypeptides are human Fabs.

[Text continues on page 2.]

More specifically, the invention is directed to the methods of cleaving single-stranded nucleic acids at chosen locations, the cleaved nucleic acids encoding, at least in part, the peptides, polypeptides or proteins displayed on the genetic packages of, and/or expressed in, the libraries of the invention.

In a preferred embodiment, the genetic packages are filamentous phage or phagemids or yeast.

The present invention further relates to vectors for displaying and/or expressing a diverse family of peptides, polypeptides or proteins.

The present invention further relates to methods of screening the libraries of the invention and to the peptides, polypeptides and proteins identified by such screening.

BACKGROUND OF THE INVENTION

It is now common practice in the art to prepare libraries of genetic packages that display, express or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. In many common libraries, the peptides, polypeptides or proteins are related to antibodies. Often, they are Fabs or single chain antibodies.

In general, the DNAs that encode members of the families to be displayed and/or expressed must be amplified before they are cloned and used to display and/or express the desired member. Such amplification typically makes use of forward and backward primers.

Such primers can be complementary to sequences native to the DNA to be amplified or complementary to oligonucleotides attached at the 5' or 3' ends of that DNA. Primers that are complementary to sequences native to the DNA to be amplified are disadvantaged in that they bias the members of the families to be displayed. Only those members that contain a sequence in the native DNA that is substantially complementary to the primer will be amplified. Those that do not will be absent from the family. For those members that are amplified, any diversity within the primer region will be suppressed.

For example, in European patent 368,684 Bl, the primer that is used is at the 5' end of the VH region of an antibody gene. It anneals to a sequence region in the native DNA that is said to be "sufficiently well conserved" within a single species. Such primer will bias the members amplified to those having this "conserved" region. Any diversity within this region is extinguished.

It is generally accepted that human antibody genes arise through a process that involves a combinatorial selection of V and J or V, D, and J followed by somatic mutations. Although most diversity occurs in the Complementary Determining Regions (CDRs) , diversity also occurs in the more conserved Framework Regions (FRs) and at least some of this diversity confers or enhances specific binding to antigens (Ag). As a consequence, libraries should contain as much of the CDR and FR diversity as possible.

To clone the amplified DNAs of the peptides, polypeptides or proteins that they encode for display on a genetic package and/or for expression, the DNAs must be cleaved to produce appropriate ends for ligation to a vector. Such cleavage is generally effected using restriction endonuclease recognition sites carried on the primers. When the primers are at the 5' end of DNA produced from reverse transcription of RNA, such restriction leaves deleterious 5' untranslated regions in the amplified DNA. These regions interfere with expression of the cloned genes and thus the display of the peptides, polypeptides and proteins coded for by them.

SUMMARY OF THE INVENTION

Embodiments of this invention provide novel methods for constructing libraries that display, express or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. These methods are not biased toward DNAs that contain native sequences that are complementary to the primers used for amplification. They also enable any sequences that may be deleterious to expression to be removed from the amplified DNA before cloning and displaying and/or expressing.

Other embodiments of this invention provide a method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the steps of: (i) contacting the nucleic acid with a singlestranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially singlestranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

Further embodiments of this invention provide an alternative method for cleaving single-stranded nucleic acid sequences at a desired location, the method comprising the step's of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the singlestranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the doublestranded region of the oligonucleotide having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially singlestranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow1 the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

In alternative embodiments, the restriction endonuclease recognition site is not initially located in the doublestranded part of the oligonucleotide. Instead, it is part of an amplification primer, which primer is complementary to the doublestranded region of the oligonucleotide. On amplification of the DNA-partially double-stranded combination, the restriction endonuclease recognition site carried on the primer becomes part of the DNA. It can then be used to cleave the DNA.

Preferably, the restriction endonuclease recognition site is that of a Type II-S restriction endonuclease whose cleavage site is located at a known distance from its recognition site.

Other embodiments of the present invention provide a method of capturing DNA molecules that comprise a member of a diverse family of DNAs and collectively comprise at least a portion of the diversity of the family. These DNA molecules in single-stranded form have been cleaved by one of the methods of this invention. This method involves ligating the individual single-stranded DNA members of the family to a partially duplex DNA complex. The method comprises the steps of: (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper reading frame for expression and containing a restriction endonuclease recognition site 5' of those sequences; and (ii) cleaving the partially double-stranded oligonucleotide sequence solely at the restriction endonuclease cleavage site contained within the doublestranded region of the partially double-stranded oligonucleotide .

As before, the restriction endonuclease recognition site need not be located in the double-stranded portion of the oligonucleotide. Instead, it can be introduced on amplification with an amplification primer that is used to amplify the DNA-partially double-stranded oligonucleotide combination.

Other embodiments of this invention provide libraries, that display, express or comprise a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least part of the diversity of the family, using the methods and DNAs described above.

Other embodiments· of this invention provide screening of these libraries to identify useful peptides, polypeptides and proteins and to use those substances in human therapy. ' A definition of a specific embodiment of the invention claimed herein follows.

In a broad format., the invention provides a vector comprising; (i) a nucleic sequence encoding an antibody variable region linked to a fragment of a wild-type pill anchor, which does not mediate infection of phage particles; and (ii) a wild-type gene III, wherein the nucleic acid sequence encoding the fragment of the wild-type pill anchor differs from the wild-type gene III.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of various methods that may be employed to amplify VH genes without using primers specific for VH sequences . FIG. 2 is a schematic of various methods that may be employed to amplify VL genes 'without using primers specific for VL sequences . FIG. 3 is a schematic of RACE amplification of antibody heavy and light chains. FIG, 4 depicts gel analysis of amplification products obtained after the primary PCR reaction from 4 different patient samples. FIG. 5 depicts gel analysis of cleaved kappa DMA from

Example 2. FIG. 6 depicts gel analysis of extender-cleaved kappa DNA from Example 2.

[Text continues on page 9.] FIG. 7 depicts gel analysis of the PCR product from the extender-kappa amplification from Example 2. FIG. 8 depicts gel analysis of purified PCR product from the extender-kappa amplification from Example 2. FIG. 9 depicts gel analysis of cleaved and ligated kappa light chains from Example 2. FIG. 10 is a schematic of the design for CDR1 and CDR2 synthetic diversity. FIG. 11 is a schemaitc of the cloning schedule for construction of the heavy chain repertoire . FIG. 12 is a schematic of the cleavage and ligation of the antibody light chain. FIG. 13 depicts gel analysis of cleaved and ligated lambda light chains from Example 4. FIG. 14 is a schematic of the cleavage and ligation of the antibody heavy chain. FIG. 15 depicts gel analysis of cleaved and ligated lambda light chains from Example 5. FIG. 16 is a schematic of a phage display vector. FIG. 17 is a schematic of a Fab cassette. FIG. 18 is a schematic of a process for incorporating fixed FR1 residues in an antibody lambda sequence. FIG. 19 is a schematic of a process for incorporating fixed FR1 residues in an antibody kappa sequence . FIG. 20 is a schematic of a process for incorporating fixed FR1 residues in an antibody heavy chain sequence.

TERMS

In this application, the following terms and abbreviations are used:

Sense strand The upper strand of ds DNA as usually written. In the sense strand, 5'-ATG-3' codes for Met.

Antisense strand The lower strand of ds DNA as usually written. In the antisense strand, 3'-TAC-5' would correspond to a Met codon in the sense strand.

Forward primer A "forward" primer is complementary to a part of the sense strand and primes for synthesis of a new antisense-strand molecule. "Forward primer" and "lower-strand primer" are equivalent.

Backward primer A "backward" primer is complementary to a part of the antisense strand and primes for synthesis of a new sense-strand molecule. "Backward primer" and "top-strand primer" are equivalent.

Bases Bases are specified either by their position in a vector or gene as their position within a gene by codon and base. For example, "89.1" is the first base of codon 89, 89.2 is the second base of codon 89.

Sv Streptavidin

Ap Ampicillin apR A gene conferring ampicillin resistance. RERS Restriction endonuclease recognition site . RE Restriction endonuclease -

cleaves preferentially at RERS ORE Universal restriction endonuclease

Functionally complementary Two sequences are sufficiently complementary so as to anneal under the chosen conditions. AA Amino acid PCR Polymerization chain reaction GLGs Germline genes

Ab Antibody: an immunoglobin.

The term also covers any protein having a binding domain which is homologous to an immunoglobin binding domain. A few examples of antibodies within this definition are, inter alia, immunoglobin isotypes and the Fab, F(ab1)2, scfv, Fv, dAb and Fd fragments.

Fab Two chain molecule comprising an Ab light chain and part of a heavy-chain. SCFv A single-chain Ab comprising

either VH::linker::VL or VL:: linker::VH w.t. Wild type HC Heavy chain LC Light chain VK A variable domain of a Kappa light chain. VH A variable domain of a heavy chain. VL A variable domain of a lambda light chain.

In this application when it is said that nucleic acids are cleaved solely at the cleavage site of a restriction endonuclease, it should be understood that minor cleavage may occur at random, e.g.f at nonspecific sites other than the specific cleavage site that is characteristic of the restriction endonuclease. The skilled worker will recognize that such nonspecific, random cleavage is the usual occurrence. Accordingly, "solely at the cleavage site" of a restriction endonuclease means that cleavage occurs preferentially at the site characteristic of that endonuclease.

As used in this application and claims, the term "cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide" includes cleavage sites formed by the single-stranded portion of the partially doublestranded ologonucleotide duplexing with the singlestranded DNA, cleavage sites in the double-stranded portion of the partially double-stranded oligonucleotide, and cleavage sites introduced by the amplification primer used to amplify the singlestranded DNA-partially double-stranded oligonucleotide combination.

In the two methods of this invention for preparing single-stranded nucleic acid sequences, the first of those cleavage sites is preferred. In the methods of this invention for capturing diversity and cloning a family of diverse nucleic acid sequences, the latter two cleavage sites are preferred.

In this application, all references referred to are specifically incorporated by reference.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The nucleic acid sequences that are useful in the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins displayed, or expressed in or comprising the libraries of this invention, may be native, synthetic or a combination thereof. They may be mRNA, DNA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies. Most preferably, they encode Fabs .

The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes. Preferably, it is introduced into CDR1 and CDR2 of immunoglobulins. Preferably, natural diversity is captured in the CDR3 regions of the immunoglogin genes of this invention from B cells. Most preferably, the nucleic acids of this invention comprise a population of immunoglobin genes that comprise synthetic diversity in at least one, and more preferably both of the CDR1 and CDR2 and diversity in CDR3 captured from B cells.

Synthetic diversity may be created, for example, through the use of TRIM technology (U.S. 5,869,644). TRIM technology allows control over exactly which amino-acid types are allowed at · variegated positions and in what proportions. In TRIM technology, codons to be diversified are synthesized using mixtures of trinucleotides. This allows any set of amino acid types to be included in any proportion.

Another alternative that may be used to generate diversified DNA is mixed oligonucleotide synthesis. With TRIM technology, one could allow Ala and Trp. With mixed oligonucleotide synthesis, a mixture that included Ala and Trp would also necessarily include Ser and Gly. The amino-acid types allowed at the variegated positions are picked with reference to the structure of antibodies, or other peptides, polypeptides or proteins of the family, the observed diversity in germline genes, the observed somatic mutations frequently observed, and the desired areas and types of variegation.

In a preferred embodiment of this invention, the nucleic acid sequences for at least one CDR or other region of the peptides, polypeptides or proteins of the family are cDNAs produced by reverse transcription from mRNA. More preferably, the mRNAs are obtained from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells (such as B-lymphocytes or plasma cells) that express members of naturally diverse sets of related genes. More preferable, the mRNAs encode a diverse family of antibodies. Most preferably, the mRNAs are obtained from patients suffering from at least one autoimmune disorder or cancer. Preferably, mRNAs containing a high diversity of autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis are used.

In a preferred embodiment of this invention, the cDNAs are produced from the mRNAs using reverse transcription. In this preferred embodiment, the mRNAs are separated from the cell and degraded using standard methods, such that only the full length (i.e., capped) mRNAs remain. The cap is then removed and reverse transcription used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., HJ de Haard et al.,

Journal of Biological Chemistry, 274(26):18218-30 (1999). In the preferred embodiment of this invention where the mRNAs encode antibodies, primers that are complementary to the constant regions of antibody genes may be used. Those primers are useful because they do not generate bias toward subclasses of antibodies. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes) . Alternatively, sequences complementary to the primer may be attached to the termini of the antisense strand.

In one preferred embodiment of this invention, the reverse transcriptase primer may be biotinylated, thus allowing the cDNA product to be immobilized on streptavidin (Sv) beads. Immobilization can also be effected using a primer labeled at the 5' end with one of a) free amine group, b) thiol, c) carboxylic acid, or d) another group not found in DNA that can react to form a strong bond to a known partner on an insoluble medium. If, for example, a free amine (preferably primary amine) is provided at the 5' end of a DNA primer, this amine can be reacted with carboxylic acid groups on a polymer bead using standard amideforming chemistry. If such preferred immobilization is used during reverse transcription, the top strand RNA is degraded using well-known enzymes, such as a combination of RNAseH and RNAseA, either before or after immobilization.

The nucleic acid sequences useful in the methods of this invention are generally amplified before being used to display and/or express the peptides, polypeptides or proteins that they encode. Prior to amplification, the single-stranded DNAs may be cleaved using either of the methods described before. Alternatively, the single-stranded DNAs may be amplified and then cleaved using one of those methods.

Any of the well known methods for amplifying nucleic acid sequences may be used for such amplification. Methods that maximize, and do not bias, diversity are preferred. In a preferred embodiment of this invention where the nucleic acid sequences are derived from antibody genes, the present invention preferably utilizes primers in the constant regions of the heavy and light chain genes and primers to a synthetic sequence that are attached at the 5' end of the sense strand. Priming at such synthetic sequence avoids the use of sequences within the variable regions of the antibody genes. Those variable region priming sites generate bias against V genes that are either of rare subclasses or that have been mutated at the priming sites'. This bias is partly due to suppression of diversity within the primer region and partly due to lack of priming when many mutations are present in the region complementary to the primer. The methods disclosed in this invention have the advantage of not biasing the population of amplified antibody genes for particular V gene types.

The synthetic sequences may be attached to the 5' end of the DNA strand by various methods well known for ligating DNA sequences together. RT CapExtention is one preferred method.

In RT CapExtention (derived from Smart PCRiTM)) , a short overlap (5 ' -. . . GGG-3 ' in the upper-strand primer (USP-GGG) complements 3'-CCC....5' in the lower strand) and reverse transcriptases are used so that the reverse complement of the upper-strand primer is attached to the lower strand. FIGs. 1 and 2 show schematics to amplify VH and VL genes using RT CapExtention. FIG. 1 shows a schematic of the amplification of VH genes. FIG. 1,

Panel A shows a primer specific to the poly-dT region of the 3' UTR priming synthesis of the first, lower strand. Primers that bind in the constant region are also suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3' terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence.

Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that replicates the 5' end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. Panel E shows immobilized doublestranded (ds) cDNA obtained by using a 5'-biotinylated top-strand primer. FIG. 2 shows a similar schematic for . amplification of VL genes. FIG. 2, Panel A shows a primer specific to the constant region at or near the 3' end priming synthesis of the first, lower strand.

Primers that bind in the poly-dT region are also suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3' terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that replicates the 5' end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. The bottom-strand primer also contains a useful restriction endonuclease site, such as Ascl. Panel E shows immobilized ds cDNA obtained by using a 5'-biotinylated top-strand primer.

In FIGs. 1 and 2, each V gene consists of a 5' untranslated region (UTR) and a secretion signal, followed by the variable region, followed by a constant region, followed by a 3' untranslated region (which typically ends in poly-A). An initial primer for reverse transcription may be complementary to the constant region or to the poly A segment of the 3'-UTR. For human heavy-chain genes, a primer of 15 T is preferred. Reverse transcriptases attach several C residues to the 3' end of the newly synthesized DNA. RT CapExtention exploits this feature. The reverse transcription reaction is first run with only a lower-strand primer. After about 1 hour, a primer ending in GGG (USP-GGG) and more RTase are added. This causes the lower-strand cDNA to be extended by the reverse complement of the USP-GGG up to the final GGG. Using one primer identical to part of the attached synthetic sequence and a second primer complementary to a region of known sequence at the 3' end of the sense strand, all the V genes are amplified irrespective of their V gene subclass.

In another preferred embodiment, synthetic sequences may be added by Rapid Amplification of cDNA Ends (RACE) (see Frohman, M.A., Dush, M.K., &amp; Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA (85): 8998-9002). FIG. 1 shows a schematic of RACE amplification of antibody heavy and light chains.

First, mRNA is selected by treating total or poly(A+) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules that have them such as ribosomal RNA, fragmented mRNA, tRNA and genomic DNA. Full length mRNA (containing a protective 7-methyl cap structure) is uneffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from full length mRNAs leaving a 5'-monophosphate group. Next, a synthetic RNA adaptor is ligated to the RNA population, only molecules which have a 5-phosphate (uncapped, full length mRNAs) will accept the adaptor. Reverse trascriptase reactions using an oligodT primer, and nested PCR (using one adaptor primer (located in the 5' synthetic adaptor) and one primer for the gene) are then used to amplify the desired transcript.

In a preferred embodiment of this invention, the upper strand or lower strand primer may be also biotinylated or labeled at the 5' end with one of a) free amino group, b) thiol, c) carboxylic acid and d) another group not found in DNA that can react to form a strong bond to a known partner as an insoluble medium. These can then be used to immobilize the labeled strand after amplification. The immobilized DNA can be either single or double-stranded.

After amplification (using e.g., RT CapExtension or RACE), the DNAs of this invention are rendered single-stranded. For example, the strands can be separated by using a biotinylated primer, capturing the biotinylated product on streptavidin beads, denaturing the DNA, and washing away the complementary strand. Depending on which end of the captured DNA is wanted, one will choose to immobilize either the upper (sense) strand or the lower (antisense) strand.

To prepare the single-stranded amplified DNAs for cloning into genetic packages so as to effect display of, or for expression of, the peptides, polypeptides or proteins encoded, at least in part, by those DNAs, they must be manipulated to provide ends suitable for cloning and display and/or expression. In particular, any 5' untranslated regions and mammalian signal sequences must be removed and replaced, in frame, by a suitable signal sequence that functions in the display or expression host. Additionally, parts of the variable domains (in antibody genes) may be removed and replaced by synthetic segments containing synthetic diversity. The diversity of other gene families may likewise be expanded with synthetic diversity.

According to the methods of this invention, there are two ways to manipulate the single-stranded DNAs for display and/or expression. The first method comprises the steps of: (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

In this first method, short oligonucleotides are annealed to the single-stranded DNA so that restriction endonuclease recognition sites formed within the now locally double-stranded regions of the DNA can be cleaved. In particular, a recognition site that occurs at the same position in a substantial fraction of the single-stranded DNAs is identical.

For antibody genes, this can be done using a catalog of germline sequences. See, e.g., "http://www.mrc-cpe. cam.ac.uk/imt-doc/restricted/ok.htm 1." Updates can be obtained from this site under the heading "Amino acid and nucleotide sequence alignments." For other families, similar comparisons exist and may be used to select appropriate regions for cleavage and to maintain diversity.

For example, Table 1 depicts the DNA sequences of the FR3 regions of the 51 known human VH germline genes. In this region, the genes contain restriction endonuclease recognition sites shown in Table 2. Restriction endonucleases that cleave a large fraction of germline genes at the same site are preferred over endonucleases that cut at a variety of sites. Furthermore, it is preferred that there be only one site for the restriction endonucleases within the region to which the short oligonucleotide binds on the single-stranded DNA, e.g., about 10 bases on either side of the restriction endonuclease recognition site.

An enzyme that cleaves downstream in FR3 is also more preferable because it captures fewer mutations in the framework. This may be advantageous is some cases. However, it is well known that framework mutations exist and confer and enhance antibody binding. The present invention, by choice of appropriate restriction site, allows all or part of FR3 diversity to be captured. Hence, the method also allows extensive diversity to be captured.

Finally, in the methods of this invention restriction endonucleases that are active between about 37°C and about 75°C are used. Preferably, restriction endonucleases that are active between about 45°C and about 75°C may be used. More preferably, enzymes that are active above 50°C, and most preferably active about 55°C, are used. Such temperatures maintain the nucleic acid sequence to be cleaved in substantially singlestranded form.

Enzymes shown in Table 2 that cut many of the heavy chain FR3 germline genes at a single position include: Maelll(2404), Tsp45I(2104), Hphl(4405),

BsaJI(23Θ65) , Alul (23047), BlpI(21048), Ddel(29658), Bglll(10661) , MslI(44072), BsiEI (23Θ74) , Eael(23674), EagI(23074), Haelll (25075), Bst4CI(51086) ,

BpyCH4III(51086) , Hint I(3802), Aflyl(1802), Pie1(1802), Mnll(31067) , BpyCH4V(21044), BsmAI(16011) , Bpml(19012), Xmnl(12030), and SacI(11051). (The notation used means, for example, that BsmAI cuts 16 of the FR3 germline genes with a restriction endonuclease recognition site beginning at base 11 of FR3.)

For cleavage of human heavy chains in FR3, the preferred restriction endonucleases are: Bst4CI (or Taal or BpyCH4III), BlpI, BpyCH4V, and Ms 11. Because ACNGT (the restriction endonuclease recognition site for Bst4Cl, Taal, and BpyCH4III) is found at a consistent site in all the human FR3 germline genes, one of those enzymes is the most preferred for capture of heavy chain CDR3 diversity. BlpI and HpyCH4V are complementary. BlpI cuts most members of the VH1 and VH4 families while HpyCH4V cuts most members of the VH3, VH5, VH6, and VH7 families. Neither enzyme cuts VH2s, but this is a very small family, containing only three members. Thus, these enzymes may also be used in preferred embodiments of the methods of this invention.

The restriction endonucleases HpyCH4III, Bst4CI, and Taal all recognize 5'-ACnGT-3' and cut upper strand DNA after n and lower strand DNA before the base complementary to n. This is the most preferred restriction endonuclease recognition site for this method on human heavy chains because it is found in all germline genes. Furthermore, the restriction endonuclease recognition region (ACnGT) matches the second and third bases of a tyrosine codon (tay) and the following cysteine codon (tay) as shown in Table 3. These codons are highly conserved, especially the cysteine in mature antibody genes.

Table 4 E shows the distinct oligonucleotides of length 22 {except the last one which is of length 20) bases. Table 5 C shows the analysis of 1617 actual heavy chain antibody genes. Of these, 1511 have the site and match one of the candidate oligonucleotides to within 4 mismatches. Eight oligonucleotides account for most of the matches and are given in Table 4 F.l. The 8 oligonucleotides are very similar so that it is likely that satisfactory cleavage will be achieved with only one oligonucleotide (such as H43.77.97.1-02#1) by adjusting temperature, pH, salinity, and the like. One or two oligonucleotides may likewise suffice whenever the germline gene sequences differ very little and especially if they differ very little close to the restriction endonuclease recognition region to be cleaved. Table 5 D shows a repeat analysis of 1617 actual heavy chain antibody genes using only the 8 chosen oligonucleotides. This shows that 1463 of the sequences match at least one of the oligonucleotides to within 4 mismatches and have the site as expected.

Only 7 sequences have a second HpyCH4III restriction endonuclease recognition region in this region.

Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human heavy chains.

Cleavage in FR1 allows capture of the entire CDR diversity of the heavy chain.

The germline genes for human heavy chain FR1 are shown in Table 6. Table 7 shows the restriction endonuclease recognition sites found in human germline genes FRls. The preferred sites are Bsgl(GTGCAG;39@4), BsoFI(GCngc;4306,1109,203,1012),

Tsel(Gcwgc;43Θ6,1109,203,1012),

MspAlI(CMGckg;4607,201), PvuII{CAGctg;4607,201),

AluI(AGct;48082Θ2), Ddel(Ctnag; 22052, 9048),

Hphl(tcacc;2208O), BssKI(Nccngg;35039, 2040),

BsaJI(Ccnngg;32040,2041), BstNI(CCwgg;33040),

ScrFI(CCngg;35040,2041), Bco0109I(RGgnccy;22046, 11043), Sau96I{Ggncc;23047,11044),

Avail(Ggwcc;23047,4044), PpuMI(RGgwccy;22046,4043), BsmFI(gtccc;20048), ffinfI(Gantc;34016,21056,21077),

Tfil(21077), Mlyl (GAGTC;34Θ16), Mlyl(gactc;21056), and AlwttI(CAGnnnctg;22068). The more preferred sites are MspAl and PvuII. MspAl and PvuII have 46 sites at 7-12 and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are used that do not fully cover the site at 1-6. Thus, the DNA will not be cleaved at that site. We have shown that DNA that extends 3, 4, or 5 bases beyond a PvuII-site can be cleaved efficiently.

Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human kappa light chains. Table 8 shows the human kappa FR1 germline genes and Table 9 shows restriction endonuclease recognition sites that are found in a substantial number of human kappa FR1 germline genes at consistent locations. Of the restriction endonuclease recognition sites listed, BsmAI and PflFI are the most preferred enzymes. BsmAI sites are found at base 18 in 35 of 40 germl'ine genes.

PflFI sites are found in 35 of 40 germline genes at base 12.

Another example of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of the human lambda light chain. Table 10 shows the 31 known human lambda FR1 germline gene sequences. Table 11 shows restriction endonuclease recognition sites found in human lambda FR1 'germline genes. Hinfl and Ddel are the most preferred restriction endonucleases for cutting human lambda chains in FR1.

After the appropriate site or sites for cleavage are chosen, one or more short oligonucleotides are prepared so as to functionally complement, alone or in combination, the chosen recognition site. The oligonucleotides also include sequences that flank the recognition site in the majority of the amplified genes. This flanking region allows the sequence to anneal to the single-stranded DNA sufficiently to allow cleavage by the restriction endonuclease specific for the site chosen.

The actual length and sequence of the oligonucleotide depends on the recognition site and the conditions to be used for contacting and cleavage. The length must be sufficient so that the oligonucleotide is functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location.

Typically, the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, annealing is too weak and above 30 bases there can be a loss of specificity. A preferred length is 18 to 24 bases .

Oligonucleotides of this length need not be identical complements of the germline genes. Rather, a few mismatches taken may be tolerated. Preferably, however, no more than 1-3 mismatches are allowed. Such mismatches do not adversely affect annealing of the oligonucleotide to the single-stranded DNA. Hence, the two DNAs are said to be functionally complementary.

The second method to manipulate the singlestranded DNAs of this invention for display and/or expression comprises the steps of: (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide; the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

As explained above, the cleavage site may be formed by the single-stranded portion of the partially double-stranded oligonucleotide duplexing with the single-stranded DNA, the cleavage site may be carried in the double-stranded portion of the partially doublestranded oligonucleotide, or the cleavage site may be introduced by the amplification primer used to amplify the single-stranded DNA-partially double-stranded oligonucleotide combination. In this embodiment, the first is preferred. And, the restriction endonuclease recognition site may be located in either the doublestranded portion of the oligonucleotide or introduced by the amplification primer, which is complementary to that double-stranded region, as used to amplify the combination.

Preferably, the restriction endonuclease site is that of a Type II-S restriction endonuclease, whose cleavage site is located at a known distance from its recognition site.

This second method, preferably, employs Universal Restriction Endonucleases ("URE"). UREs are partially double-stranded oligonucleotides. The single-stranded portion or overlap of the URE consists of a DNA adapter that is functionally complementary to the sequence to be cleaved in the single-stranded DNA. The double-stranded portion consists of a restriction endonuclease recognition site, preferably type II-S.

The URE method of this invention is specific and precise and can tolerate some (e.g., 1-3) mismatches in the complementary regions, i.e., it is functionally complementary to that region. Further, conditions under which the URE is used can be adjusted so that most of the genes that are amplified can be cut, reducing bias in the library produced from those genes.

The sequence of the single-stranded DNA adapter or overlap portion of the URE typically consists of about 14-22 bases. However, longer or shorter adapters may be used. The size depends on the ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used for contacting the URE and the singlestranded DNA at the temperature used for cleaving the DNA with the restriction enzyme. The adapter must be functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that the cleavage may occur at the chosen temperature and at the desired location. We prefer singe-stranded or overlap portions of 14-17 bases in length, and more preferably 18-20 bases in length.

The site chosen for cleavage using the URE is preferably one that is substantially conserved in the family of amplified DNAs. As compared to the first cleavage method of this invention, these sites do not need to be endonuclease recognition sites. However, like the first method, the sites chosen can be synthetic rather than existing in the native DNA. Such sites may be chosen by references to the -sequences of known antibodies or other families of genes. For example, the sequences of many germline genes are reported at http://www.mrc-coe. cam.ac.uk/imt-doc/restricted/ok.html. For example, one preferred site occurs near the end of FR3 — codon 89 through the second base of codon 93. CDR3 begins at codon 95.

The sequences of 79 human heavy-chain genes are also available at http://www.ncbi.nlm.nih.gov/entre2/nucleotide.html.

This site can be used to identify appropriate sequences for URE cleavage according to the methods of this invention. See, e.g., Table 12B.

Most preferably, one or more sequences are identified using these sites or other available sequence information. These sequences together are present in a substantial fraction of the amplified DNAs. For example, multiple sequences could be used to allow for known diversity in germline genes or for frequent somatic mutations. Synthetic degenerate sequences could also be used. Preferably, a sequence(s) that occurs in at least 65% of genes examined with no more than 2-3 mismatches is chosen URE single-stranded adapters or overlaps are then made to be complementary to the chosen regions. Conditions for using the UREs are determined empirically. These conditions should allow cleavage of DNA that contains the functionally complementary sequences with no more than 2 or 3 mismatches but that do not allow cleavage of DNA lacking such sequences.

As described above, the double-stranded portion of the URE includes an endonuclease recognition site, preferably a Type II-S recognition site. Any enzyme that is active at a temperature necessary to maintain the single-stranded DNA substantially in that form and to allow the single-stranded DNA adapter portion of the URE to anneal long enough to the singlestranded DNA to permit cleavage at the desired site may be used.

The preferred Type II-S enzymes for use in the URE methods of this invention provide asymmetrical cleavage of the single-stranded DNA. Among these are the enzymes listed in Table 13. The most preferred Type II-S enzyme is Fokl.

When the preferred Fokl containing URE is used, several conditions are preferably used to effect cleavage: 1) Excess of the URE over target DNA should be present to activate the enzyme. URE present only in equimolar amounts to the target DNA would yield poor cleavage of ssDNA because the amount of active enzyme available would be limiting. 2) An activator may be used to activate part of the Fokl enzyme to dimerize without causing cleavage. Examples of appropriate activators are shown in Table 14. 3) The cleavage reaction is performed at a temperature between 45°-75°C, preferably above 50°C and most preferably above 55°C.

The UREs used in the prior art contained a 14-base single-stranded segment, a 10-base stem (containing a Fokl site), followed by the palindrome of the 10-base stem. While such UREs may be used in the methods of this invention, the preferred UREs of this invention also include a segment of three to eight bases (a loop) between the Fokl restriction endonuclease recognition site containing segments. In the preferred embodiment, the stem (containing the Fokl site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these "lollipop" ORE adapters are shown in Table 15.

One example of using a URE to cleave an single-stranded DNA involves the FR3 region of human heavy chain. Table 16 shows an analysis of 840 full-length mature human heavy chains with the URE recognition sequences shown. The vast majority (718/840=0.85) will be recognized with 2 or fewer mismatches using five UREs (VHS881-1.1, VHS881-1.2, VHS881-2.1, VHS881-4.1, and VHS881-9.1). Each has a 20-base adaptor sequence to complement the germline gene, a ten-base stem segment containing a FokI site, a five base loop, and the reverse complement of the first stem segment. Annealing those adapters, alone or in combination, to single-stranded antisense heavy chain DNA and treating with FokI in the presence of, e.g., the activator FOKIact, will lead to cleavage of the antisense strand at the position indicated.

Another example of using a URE(s) to cleave a single-stranded DNA involves the FR1 region of the human Kappa light chains. Table 17 shows an analysis of 182 full-length human kappa chains for matching by the four 19-base probe sequences shown. Ninety-six percent of the sequences match one of the probes with 2 or fewer mismatches. The URE adapters shown in Table 17 are for cleavage of the sense strand of kappa chains. Thus, the adaptor sequences are the reverse complement of the germline gene sequences. The URE consists of a ten-base stem, a five base loop, the reverse complement of the stem and the complementation sequence. The loop shown here is TTGTT, but other sequences could be used. Its function is to interrupt the palindrome of the stems so that formation of a lollypop monomer is favored over dimerization. Table 17 also shows where the sense strand is cleaved.

Another example of using a URE to cleave a single-stranded DNA involves the human lambda light chain. Table 18 shows analysis of 128 human lambda light chains for matching the four 19-base probes shown. With three or fewer mismatches, 88 of 128 (69%) of the chains match one of the probes. Table 18 also shows URE adapters corresponding to these probes. Annealing these adapters to upper-strand ssDNA of lambda chains and treatment with Fokl in the presence of FOKIact at a temperature at or above 45°C will lead to specific and precise cleavage of the chains.

The conditions under which the short oligonucleotide seguences of the first method and the UREs of the second method are contacted with the single-stranded DNAs may be empirically determined.

The conditions must be such that the single-stranded DNA remains in substantially single-stranded form.

More particularly, the conditions must be such that the single-stranded DNA does not form loops that may intvrfere with its association with the oligonucleotide sequence or the URE or that may themselves provide sites for cleavage by the chosen restriction endonuclease.

The effectiveness and specificity of short oligonucleotides (first method) and UREs (second method) can be adjusted by controlling the concentrations of the URE adapters/oligonucleotides and substrate DNA, the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and formamide), the concentration of the restriction endonuclease (e. g., FokI) , and the time of the digestion. These conditions can be optimized with synthetic oligonucleotides having: 1) target germline gene sequences, 2) mutated target gene sequences, or 3) somewhat related non-target sequences. The goal is to cleave most of the target sequences and minimal amounts of non-targets.

In accordance with this invention, the single—stranded DNA is maintained in substantially that form using a temperature between about 37°C and about 75°C. Preferably, a temperature between about 45°C and about 75°C is used. More preferably, a temperature between 50°C and 60°C, most preferably between 55 C and 60°C, is used. These temperatures are employed both when contacting the DNA with the oligonucleotide or URE and when cleaving the DNA using the methods of this invention.

The two cleavage methods of this invention have several advantages. The first method allows the individual members of the family of single-stranded DNAs to be cleaved preferentially at one substantially conserved endonuclease recognition site. The method also does not require an endonuclease recognition site to be built into the reverse transcription or amplification primers. Any native or synthetic site in the family can be used.

The second method has both of these advantages. In addition, the preferred URE method allows the single-stranded DNAs to be cleaved at positions where no endonuclease recognition site naturally occurs or has been synthetically constructed.

Most importantly, both cleavage methods permit the use of 5' and 3' primers so as to maximize diversity and then cleavage to remove unwanted or deleterious sequences before cloning, display and/or expression.

After cleavage of the amplified DNAs using one of the methods of this invention, the DNA is prepared for cloning, display and/or expression. This is done by using a partially duplexed synthetic DNA adapter, whose terminal sequence is based on the specific cleavage site at which the amplified DNA has been cleaved.

The synthetic DNA is designed such that when it is ligated to the cleaved single-stranded DNA in proper reading frame so that the desired peptide, polypeptide or protein can be displayed on the surface of the genetic package and/or expressed. Preferably, the double-stranded portion of the adapter comprises the sequence of several codons that encode the amino acid sequence characteristic of the family of peptides, polypeptides or proteins up to the cleavage site. For human heavy chains, the amino acids of the 3-23 framework are preferably used to provide the sequences required for expression of the cleaved DNA.

Preferably, the double-stranded portion of the adapter is about 12 to 100 bases in length. More preferably, about 20 to 100 bases are used. The double-standard region of the adapter also preferably contains at least one endonuclease recognition site useful for cloning the DNA into a suitable display and/or expression vector {or a recipient vector used to archive the diversity). This endonuclease restriction site may be native to the germline gene sequences used to extend the DNA sequence. It may be also constructed using degenerate sequences to the native germline gene sequences. Or, it may be wholly synthetic.

The single-stranded portion of the adapter is complementary to the region of the cleavage in the single-stranded DNA. The overlap can be from about 2 bases up to about 15 bases. The longer the overlap, the more efficient the ligation is likely to be. A preferred length for the overlap is 7 to 10. This allows some mismatches in the region so that diversity in this region may be captured.

The single-stranded region or overlap of the partially duplexed adapter is advantageous because it allows DNA cleaved at the chosen site, but not other fragments to be captured. Such fragments would contaminate the library with genes encoding sequences that will not fold into proper antibodies and are likely to be non-specifically sticky.

One illustration of the use of a partially duplexed adaptor in the methods of this invention involves ligating such adaptor to a human FR3 region that has been cleaved, as described above, at 5'-ACnGT-3' using HpyCH4III, Bst4CI or Taal.

Table 4 F.2 shows the bottom strand of the double-stranded portion of the adaptor for ligation to the cleaved bottom-strand DNA. Since the HpyCH4III-Site is so far to the right (as shown in Table 3), a sequence that includes the Aflll-site as well as the Xbal site can be added. This bottom strand portion of the partially-duplexed adaptor, H43.XAExt, incorporates both Xbal and AfUI-sites. The top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments opposite the XJbal and Aflll-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt. H43AExt contains only the Aflllsite and is to be used with the top strands H43.ABrl and H43.ABr2 (which have intentional alterations to destroy the Aflllsite) .

After ligation, the desired, captured DNA can be PCR amplified again, if desired, using in the preferred embodiment a primer to the downstream constant region of the antibody gene and a primer to part of the double-standard region of the adapter. The primers may also carry restriction endonuclease sites for use in cloning the amplified DNA.

After ligation, and perhaps amplification, of the partially double-stranded adapter to the singlestranded amplified DNA, the composite DNA is cleaved at chosen 5' and 3' endonuclease recognition sites.

The cleavage sites useful for cloning depend on the phage or phagemid or other vectors into which the cassette will be inserted and the available sites in the antibody genes. Table 19 provides restriction endonuclease data for 75 human light chains. Table 20 shows corresponding data for 79 human heavy chains. In each Table, the endonucleases are ordered by increasing frequency of cutting. In these Tables, Nch is the number of chains cut by the enzyme and Ns is the number of sites (some chains have more than one site) .

From this analysis, Sfil, Notl, Aflll, ApaLJ, and AscI are very suitable. Sfil and Not I are preferably used in pCESl to insert the heavy-chain display segment. ApaLI and AscI are preferably used in pCESl to insert the light-chain display segment.

BstΕΙΙ-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/79 (68%) of heavy-chain genes contain a BstEll-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEll-Site. An alternative to using BstEII is to cleave via UREs at the end of JH and ligate to a synthetic oligonucleotide that encodes part of CHI.

One example of preparing a family of DNA sequences using the methods of this invention involves capturing human CDR 3 diversity. As described above, mRNAs from various autoimmune patients are reverse transcribed into lower strand cDNA. After the top strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide used to cleave the cDNA upstream of CDR3. A partially duplexed synthetic DNA adapter is then annealed to the DNA and the DNA is amplified using a primer to the adapter and a primer to the constant region (after FR4). The DNA is then cleaved using BstEII (in FR4) and a restriction endonuclease appropriate to the partially doublestranded adapter [e.g., Xbal and Aflll (in FR3)). The DNA is then ligated into a synthetic VH skeleton such as 3-23.

One example of preparing a single-stranded DNA that was cleaved using the URE method involves the human Kappa chain. The cleavage site in the sense strand of this chain is depicted in Table 17. The oligonucleotide kapextURE is annealed to the oligonucleotides (kaBROlUR, kaBR02UR, kaBR03UR, and kaBR04UR) to form a partially duplex DNA. This DNA is then ligated to the cleaved soluble kappa chains. The ligation product is then amplified using primers kapextUREPCR and CKForeAsc (which inserts a AscI site after the end of C kappa). This product is then cleaved with ApaLI and AscI and ligated to similarly cut recipient vector.

Another example involves the cleavage of lambda light chains, illustrated in Table 18. After cleavage, an extender (ON_LamExl33) and four bridge oligonucleotides (ON_LamBl-133, ON_LamB2-133, ON_LamB3-133, and ON_LamB4-l33) are annealed to form a partially duplex DNA. That DNA is ligated to the cleaved lambda-chain sense strands. After ligation, the DNA is amplified with ON_Laml33pcr and a forward primer specific to the lambda constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130).

In human heavy chains, one can cleave almost all genes in FR4 (downstream, i.e., toward the 3’ end of the sense strand, of CDR3) at a BstEII-Site that occurs at a constant position in a very large fraction of human heavy-chain V genes. One then needs a site in FR3, if only CDR3 diversity is to be captured, in FR2,e if CDR2 and CDR3 diversity is wanted, or in FR1, if all the CDR diversity is wanted. These sites are preferably inserted as part of the partially doublestranded adaptor.

The preferred process of this invention is to provide recipient vectors (e.g., for display and/or expression) having sites that allow cloning of either light or heavy chains. Such vectors are well known and widely used in the art. A preferred phage display vector in accordance with this invention is phage MALIA3. This displays in gene III. The sequence of the phage MALIA3 is shown in Table 21A (annotated) and Table 21B (condensed).

The DNA encoding the selected regions of the light or heavy chains can be transferred to the vectors using endonucleases that cut either light or heavy chains only very rarely. For example, light chains may be captured with ApaLI and AscI. Heavy-chain genes are preferably cloned into a recipient vector having SfiI, Ncol, Xbal, A fill, BstEU, Apal, and No tl sites. The light chains are preferably moved into the library as ApaLI-AscI fragments. The heavy chains are preferably moved into the library as Sfil-Notl fragments.

Most preferably, the display is had on the surface of a derivative of M13 phage. The most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred vector is stable against rearrangement under the growth conditions used to amplify phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a phagemid vector (e.g., pCESl) that displays and/or expresses the peptide, polypeptide or protein. Such vectors may also be used to store the diversity for subsequent display and/or expression using other vectors or phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a yeast vector.

In another embodiment, the mode of display may be through a short linker to anchor domains -- one possible anchor comprising the final portion of M13 III {"Illstump") and a second possible anchor being the full length III mature protein.

The Illstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. Ill proteins are present the phage is unlikely to delete the antibody genes and phage that do delete these segments receive only a very small growth advantage.

For each of the anchor domains, the DNA encodes the w.t. AA sequence, but differs from the w.t. DNA sequence to a very high extent. This will greatly reduce the potential for homologous recombination between the anchor and the w.t. gene that is also present (see Example 6).

Most preferably, the present invention uses a complete phage carrying an antibiotic-resistance gene (such as an ampicillin-resistance gene) and the display cassette. Because the w.t. Hi and possibly viii genes are present, the w.t. proteins are also present. The display cassette is transcribed from a regulatable promoter (e.g., Plscz) · Use of a regulatable promoter allows control of the ratio of the fusion display gene to the corresponding w.t. coat protein. This ratio determines the average number of copies of the display fusion per phage (or phagemid) particle.

Another aspect of the invention is a method of displaying peptides, polypeptides or proteins (and particularly Fabs) on filamentous phage. In the most preferred embodiment this method displays FABs and comprises : a) obtaining a cassette capturing a diversity of segments of DNA encoding the elements:

Preg: :RBS1: :SS1: :VL: :CL: :stop: :RBS2: :SS2: :VH: :CH1: : linker: -.anchor: : stop: :, where Preg is a regulatable promoter, RBS1 is a first ribosome binding site, SSI is a signal sequence operable in the host strain, VL is a member of a diverse set of light-chain variable regions, CL is a light-chain constant region, stop is one or more stop codons, RBS2 is a second ribosome binding site, SS2 is a second signal sequence operable in the host strain, VH is a member of a diverse set of heavy-chain variable regions, CHI is an antibody heavy-chain first constant domain, linker is a sequence of amino acids of one to about 50 residues, anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons; and b) positioning that cassette within the phage genome to maximize the viability of the phage and to minimize the potential for deletion of the cassette or parts thereof.

The DNA encoding the anchor protein in the above preferred cassette should be designed to encode the same (or a closely related) amino acid sequence as is found in one of the coat proteins of the phage, but with a distinct DNA sequence. This is to prevent unwanted homologous recombination with the w.t. gene.

In addition, the cassette should be placed in the intergenic region. The positioning and orientation of the display cassette can influence the behavior of the phage.

In one embodiment of the invention, a transcription terminator may be placed after the second stop of the display cassette above (e.g., Trp). This will reduce interaction between the display cassette and other genes in the phage antibody display vector.

In another embodiment of the methods of this invention, the phage or phagemid can display and/or express proteins other than Fab, by replacing the Fab portions indicated above, with other protein genes.

Various hosts can be used the display and/or expression aspect of this invention. Such hosts are well known in the art. In the preferred embodiment, where Fabs are being displayed and/or expressed, the preferred host should grow at 30°C and be RecA (to reduce unwanted genetic recombination) and EndA (to make recovery of RF DNA easier). It is also preferred that the host strain be easily transformed by electroporation - XLl-Blue MRF' satisfies most of these preferences, but does not grow well at 30 C. XLl-Blue MRF' does grow slowly at 38°C and thus is an acceptable host. TG-1 is also an acceptable host although it is RecA+ and EndA+. XLl-Blue MRF' is more preferred for the intermediate host used to accumulate diversity prior to final construction of the library.

After display and/or expression, the libraries of this invention may be screened using well known and conventionally used techniques. The selected peptides, polypeptides or proteins may then be used to treat disease. Generally, the peptides, polypeptides or proteins for use in therapy or in pharmaceutical compositions are produced by isolating the DNA encoding the desired peptide, polypeptide or protein from the member of the library selected. That DNA is then used in conventional methods to produce the peptide, polypeptides or protein it encodes in appropriate host cells, preferably mammalian host cells, e.g., CHO cells. After isolation, the peptide, polypeptide or protein is used alone or with pharmaceutically acceptable compositions in therapy to treat disease.

EXAMPLES

Example 1: RACE amplification of heavy and light chain antibody repertoires from autoimmune patients.

Total RNA was isolated from individual blood samples (50 ml) of 11 patients using a RNAzolTM kit (CINNA/Biotecx), as described by the manufacturer. The patients were diagnosed as follows: 1. SLE and phospholipid syndrome 2. limited systemic sclerosis 3. SLE and Sjogren syndrome 4. Limited Systemic sclerosis 5. Reumatoid Arthritis with active vasculitis 6. Limited systemic sclerosis and Sjogren Syndrome 7. Reumatoid Artritis and (not active) vasculitis 8. SLE and Sjogren syndrome

9. SLE 10. SLE and (active) glomerulonephritis 11. Polyarthritis/ Raynauds Phenomen

From these 11 samples of total RNA, Poly-A+ RNA was isolated using Promega PolyATtract® mRNA Isolation kit (Promega). 250 ng of each poly-A+ RNA sample was used to amplify antibody heavy and light chains with the GeneRAacerTM kit (Invitrogen cat no. L1500-01). A schematic overview of the RACE procedure is shown in FIG. 3.

Using the general protocol of the GeneRAacer kit, an RNA adaptor was ligated to the 5'end of all mRNAs. Next, a reverse transcriptase reaction was performed in the presence of oligo(dT15) specific primer under conditions described by the manufacturer ΓΝ , in the GeneRAacer kit. 1/5 of the cDNA from the reverse transcriptase reaction was used in a 20 ul PCR reaction. For amplification of the heavy chain IgM repertoire, a forward primer based on the CHI chain of IgM [HuCmFOR] and a backward primer based on the ligated synthetic adaptor sequence [5Ά] were used. (See Table 22)

For amplification of the kappa and lambda light chains, a forward primer that contains the 3’ coding-end of the cDNA [HuCkFor and HuCLFor2+HuCLfor7] and a backward primer based on the ligated synthetic adapter sequence [5'A] was used (See Table 22).

Specific amplification products after 30 cycles of primary PCR were obtained. FIG. 4 shows the amplification products obtained after the primary PCR reaction from 4 different patient samples. 8 ul primary PCR product from 4 different patients was analyzed on a agarose gel [labeled 1,2, 3 and 4). For the heavy chain, a product of approximately 950 nt is obtained while for the kappa and lambda light chains the product is approximately 850 nt. Ml-2 are molecular weight markers. PCR products were also analyzed by DNA sequencing [10 clones from the lambda, kappa or heavy chain repertoires]. All sequenced antibody genes recovered contained the full coding sequence as well as the 5' leader sequence and the V gene diversity was the expected diversity (compared to literature data). 50 ng of all samples from all 11 individual amplified samples were mixed for heavy, lambda light or kappa light chains and used in secondary PCR reactions.

In all secondary PCRs approximately 1 ng template DNA from the primary PCR mixture was used in multiple 50 ul PCR reactions [25 cycles].

For the heavy chain, a nested biotinylated -forward primer [HuCm-Nested] was used, and a nested 5'end backward primer located in the synthetic adapter-sequence [5'NA] was used. The 5'end lower-strand of the heavy chain was biotinylated.

For the light chains, a 5'end biotinylated nested primer in the synthetic adapter was used [5'NA] in combination with a 3'end primer in the constant region of Ckappa and Clambda, extended with a sequence coding for the AscI restriction site [ kappa: HuCkForAscI, Lambda: HuCL2-FOR-ASC + HuCL7-FOR-ASC] .

[5'end Top strand DNA was biotinylated]. After gel-analysis the secondary PCR products were pooled and purified with Promega Wizzard PCR cleanup.

Approximately 25 ug biotinylated heavy chain, lambda and kappa light chain DNA was isolated from the 11 patients.

Example 2: Capturing kappa chains with BsmAI. A repertoire of human-kappa chain mRNAs was prepared using the RACE method of Example 1 from a collection of patients having various autoimmune diseases.

This Example followed the protocol of Example 1. Approximately 2 micrograms (ug) of human kappa-chain (Igkappa) gene RACE material with biotin attached to 5'-end of upper strand was immobilized as in Example 1 on 200 microliters (pL) of Seradyn magnetic beads.

The lower strand was removed by washing the DNA with 2 aliquots 200 pL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 seconds for the second aliquot. The beads were neutralized with 200 pL of 10 mM Tris (pH 7.5) 100 mM NaCl. The short oligonucleotides shown in Table 23 were added in 40 fold molar excess in 100 pL of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol pH 7.9) to the dry beads. The mixture was incubated at 95°C for 5 minutes then cooled down to 55°C over 30 minutes. Excess oligonucleotide was washed away with 2 washes of NEB buffer 3 (100 mM NaCl, 50 xnM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol pH 7.9). Ten units of BsmAI (NEB) were added in NEB buffer 3 and incubated for 1 h at 55°C. The cleaved downstream DNA was collected and purified over a Qiagen PCR purification column (FIGs. 5 and 6). FIG. 5 shows an analysis of digested kappa single-stranded DNA. Approximately 151.5 pmol of adapter was annealed to 3.79 pmol of immobilized kappa single-stranded DNA followed by digestion with 15 U of BsmAI. The supernatant containing the desired DNA was removed and analyzed by 5% polyacrylamide gel along with the remaining beads which contained uncleaved full length kappa DNA. 189 pmol of cleaved single-stranded DNA was purified for further analysis. Five percent of the original full length ssDNA remained on the beads. FIG. 6 shows an analysis of the extender -cleaved kappa ligation. 180 pmol of pre-annealed bridge/extender was ligated to 1.8 pmol of BsmAI digested single-stranded DNA. The ligated DNA was purified by Qiagen PCR purification column and analyzed on a 5% polyacrylamide gel. Results indicated that the ligation of extender to single-stranded DNA was 95% efficient. A partially double-stranded adaptor was prepared using the oligonucleotide shown in Table 23. The adaptor was added to the single-stranded DNA in 100 fold molar excess along with 1000 units of T4 DNA ligase and incubated overnight at 16°C. The excess oligonucleotide was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using the primers kapPCRtl and kapfor shown in Table 23 for 10 cycles with the program shown in Table 24.

The soluble PCR product was run on a gel and showed a band of approximately 700 n, as expected (FIGs. 7 and 8). The DNA was cleaved with enzymes ApaLI and AscI, gel purified, and ligated to similarly cleaved vector pCESl. FIG. 7 shows an analysis of the PCR product from the extender-kappa amplification. Ligated extender-kappa single-stranded DNA was amplified with primers specific to the extender and to the constant region of the light chain. Two different template concentrations, 10 ng versus 50 ng, were used as template and 13 cycles were used to generate approximately 1.5 ug of dsDNA as shown by 0.8% agarose gel analysis. FIG. 8 shows an analysis of the purified PCR product from the extender-kappa amplification. Approximately 5 ug of PCR amplified extender-kappa double-stranded DNA was run out on a 0.8% agarose gel, cut out, and extracted with a GFX gel purification column. By gel analysis, 3.5 ug of double-stranded DNA was prepared.

The assay for capturing kappa chains with BsmAl was repeated and produced similar results. FIG 9A shows the DNA after it was cleaved and collected and purified over a Qiagen PCR purification column. FIG. 9B shows the partially double-stranded adaptor ligated to the single-stranded DNA. This ligated material was then amplified (FIG. 9C). The gel showed a band of approximately 700 n.

Table 25 shows the DNA sequence of a kappa light chain captured by this procedure. Table 26 shows a second sequence captured by this procedure. The closest bridge sequence was complementary to the sequence 5'-agccacc-3', but the sequence captured reads 5'-Tgccacc-3’, showing that some mismatch in the overlapped region is tolerated.

Example 3: Construction of Synthetic CDR1 and CDR2 Diversity in V-3-23 VH Framework.

Synthetic diversity in Complementary Determinant Region (CDR) 1 and 2 was created in the 323 VH framework in a two step process: first, a vector containing the 3-23 VH framework was constructed; and then, a synthetic CDR 1 and 2 was assembled and cloned into this vector.

For construction of the 3-23 VH framework, 8 oligonucleotides and two PCR primers (long oligonucleotides - T0PFR1A, B0TFR1B, B0TFR2, B0TFR3, F06, B0TFR4, ON-vgCl, and 0N-vgC2 and primers - SFPRMET and BOTPCRPRIM, shown in Table 27) that overlap were designed based on the Genebank sequence of 3-23 VH framework region. The design incorporated at least one useful restriction site in each framework region, as shown in Table 27. In Table 27, the segments that were synthesized are shown as bold, the overlapping regions are underscored, and the PCR priming regions at each end are underscored. A mixture of these 8 oligos was combined at a final concentration of 2.5uM in a 20ul PCR reaction.

The PCR mixture contained 200uM dNTPs, 2.5mM MgCl2, 0.02U Pfu Turbo™ DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, and IX Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s.

The assembled 3-23 VH DNA sequence was then amplified, using 2.5ul of a 10-fold dilution from the initial PCR in lOOul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5mM MgCl2, 0.02U Pfu Turbo™ DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, IX Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of luM. The PCR program consisted of 23 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 60s. The 3-23 VH DNA sequence was digested and cloned into pCESl (phagemid vector) using the Sjfil and BstEII restriction endonuclease sites.

All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, MA and used as per the manufacturer's instructions.

Stuffer sequences (shown in Table 28 and Table 29) were introduced into pCESl to replace CDR1/CDR2 sequences (900 bases between BspEI and Xbal RE sites) and CDR3 sequences (358 bases between AflII and BstEII) prior to cloning the CDR1/CDR2 diversity. This new vector was termed pCES5 and its sequence is given in Table 29.

Having stuffers in place of the CDRs avoids the risk that a parental sequence would be overrepresented in the library. The stuffer sequences are fragments from the penicillase gene of E. coli. The CDR1-2 stuffer contains restriction sites for BglII, Bsu36I, Bell, Xcml, Ml ul, PvuII, Hpal, and Hindi, the underscored sites being unique within the vector pCES5. The stuffer that replaces CDR3 contains the unique restriction endonuclease site RsrII. A schematic representation of the design for CDR1 and CDR2 synthetic diversity is shown FIG. 10.

The design was based on the presence of mutations in DP47/3-23 and related germline genes. Diversity was designed to be introduced at the positions within CDR1 and CDR2 indicated by the numbers in FIG. 10. The diversity at each position was chosen to be one of the three following schemes: 1 = ADEFGHIKLMNPQRSTVWY; 2 = YRWVGS; 3 = PS, in which letters encode equimolar mixes of the indicated amino acids.

For the construction of the CDR1 and CDR2 diversity, 4 overlapping oligonucleotides (ON-vgCl, 0N_Brl2, ON_CD2Xba, and 0N-vgC2, shown in Table 27 and Table 30) encoding CDR1/2, plus flanking regions, were designed. A mixture of these 4 oligos was combined at a final concentration of 2.5uM in a 40ul PCR reaction. Two of the 4 oligos contained variegated sequences positioned at the CDR1 and the CDR2. The PCR mixture contained 200uM dNTPs, 2.5U Pwo DNA Polymerase (Roche), and IX Pwo PCR buffer with 2mM MgSO„. The PCR program consisted of 10 cycles at 94°C for 30s, 60°C for 30s, and 72°C for 60s. This assembled CDR1/2 DNA sequence was amplified, using 2.5ul of the mixture in lOOul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5U Pwo DNA Polymerase, IX Pwo PCR Buffer with 2mM MgS04 and 2 outside primers at a concentration of luM. The PCR program consisted of 10 cycles at 94°C for 30s, 60°C for 30s, and 72°C for 60s. These variegated sequences were digested and cloned into the 3-23 VH framework in place of the CDR1/2 stuffer.

We obtained approximately 7 X 107 independent transformants. CDR3 diversity either from donor populations or from synthetic DNA can be cloned into the vector containing synthetic CDR1 and CDR 2 diversity. A schematic representation of this procedure is shown in FIG. 11. A sequence encoding the FR-regions of the human V3-23 gene segment and CDR regions with synthetic diversity was made by oligonucleotide assembly and cloning via BspEl and Xbal sites into a vector that complements the FR1 and FR3 regions. Into this library of synthetic VH segments, the complementary VH-CDR3 sequence (top right) was cloned via Xbal an BstEll sites. The resulting cloned CH genes contain a combination of designed synthetic diversity and natural diversity (see FIG. 11) .

Example 4: Cleavage and ligation of the lambda light chains with HinfI. A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 12Δ and 12B. Approximately 2 ug of biotinylated human Lambda DNA prepared as described in Example 1 was immobilized on 200 ul Seradyn magnetic beads. The lower strand was removed by incubation of the DNA with 200 ul of 0.1 M NaOH (pH=13) for 3 minutes, the supernatant was removed and an additional washing of 30 seconds with 200 ul of 0.1 M NaOH was performed. Supernatant was removed and the beads were neutralized with 200 ul of 10 mM Tris (pH=7.5), 100 mM NaCl. 2 additional washes with 200 ul NEB2 buffer 2, containing 10 mM Tris (pH=7.9), 50 mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol, were performed. After immobilization, the amount of ssDNA was estimated on a 5% PAGE-UREA gel. ' About 0.8 ug ssDNA was recovered and incubated in 100 ul NEB2 buffer 2 containing 80 molar fold excess of an equimolar mix of ON_LamlaB7, ON_Lam2aB7, ON_Lam31B7 and ON_Lam3rB7 [each oligo in 20 fold molar excess] (see Table 31).

The mixture was incubated at 95° C for 5 minutes and then slowly cooled down to 50° C over a period of 30 minutes. Excess of oligonucleotide was washed away with 2 washes of 200 ul of NEB buffer 2. 4 U/ug of Hinf I was added and incubated for 1 hour at 50° C. Beads were mixed every 10 minutes.

After incubation the sample was purified over a Qiagen PCR purification column and was subsequently analysed on a 5% PAGE-urea gel (see FIG. 13A, cleavage was more than 70% efficient). A schematic of the ligation of the cleaved light chains is shown in FIG. 12B. A mix of bridge/extender pairs was prepared from the Brg/Ext oligo's listed in Table 31 {total molar excess 100 fold) in 1000 U of T4 DNA Ligase (NEB) and incubated overnight at 16c C. After ligation of the DNA, the excess oligonucleotide was removed with a Qiagen PCR purification column and ligation was checked on a Urea-PAGE gel (see FIG. 13B; ligation was more than 95% efficient).

Multiple PCRs were performed containing 10 ng of the ligated material in an 50 ul PCR reaction using 25 pMol ON lamPlePCR and 25 pmol of an eguimolar mix of Hu-CL2AscI/HuCL7AscI primer (see Example 1) . PCR was performed at 60° C for 15 cycles using Pfu polymerase. About 1 ug of dsDNA was recovered per PCR (see FIG. 13C) and cleaved with ApaLl and AscI for cloning the lambda light chains in pCES2.

Example 5: Capture of human heavy-chain CDR3 population. A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 14A and 14B.

Approximately 3 ug of human heavy-chain (IgM) gene RACE material with biotin attached to 5'-end of lower strand was immobilized on 300 uL of Seradyn magnetic beads. The upper strand was removed by washing the DNA with 2 aliquots 300 uL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 seconds for the second aliquot. The beads were neutralized with 300 uL of 10 mM Tris (pH 7.5) 100 mM NaCl. The REdaptors (oligonucleotides used to make single-stranded DNA locally double-stranded) shown in Table 32 were added in 30 fold molar excess in 200 uL of NEB buffer 4 (50 mM Potasium Acetate, 20 mM Tris-Acetate, 10 mM Magnesuim Acetate, 1 mM dithiothreitol pH 7.9) to the dry beads. The REadaptors were incubated with the single-stranded DNA at 80 °C for 5 minutes then cooled down to 55 °C over 30 minutes. Excess REdaptors were washed away with 2 washes of NEB buffer 4. Fifteen units of HpyCH4III (NEB) were added in NEB buffer 4 and incubated for 1 hour at 55 °C. The cleaved downstream DNA remaining on the beads was removed from the beads using a Qiagen Nucleotide removal column (see FIG. 15).

The Bridge/Extender pairs shown in Table 33 were added in 25 molar excess along with 1200 units of T4 DNA ligase and incubated overnight at 16 °C. Excess Bridge/Extender was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using primers H43.XAExtPCR2 and Hucumnest shown in Table 34 for 10 cycles with the program shown in Table 35.

The soluble PCR product was run on a gel and showed a band of approximately 500 n, as expected (see FIG. 15B) . The DNA was cleaved with enzymes Sfil and Notl, gel purified, and ligated to similarly cleaved vector PCES1.

Example 6: Description of Phage Display Vector CJRA05, a member of the library built in vector DY3F7.

Table 36 contains an annotated DNA sequence of a member of the library, CJRA05, see FIG. 16. Table 36 is to be read as follows: on each line everything that follows an exclamation mark "!" is a comment. All occurrences of A, C, G, and T before "!" are the DNA sequence. Case is used only to show that certain bases constitute special features, such as restriction sites, ribosome binding sites, and the like, which are labeled below the DNA. CJRA05 is a derivative of phage DY3F7, obtained by cloning an ApaLI to Notl fragment into these sites in DY3F31. DY3F31 is like DY3F7 except that the light chain and heavy chain genes have been replaced by "stuffer" DNA that does not code for any antibody. DY3F7 contains an antibody that binds streptavidin, but did not come from the present library.

The phage genes start with gene ii and continue with genes x, v, vii, ix, viii, iii, vi, i, and iv. Gene iii has been slightly modified in that eight codons have been inserted between the signal sequence and the mature protein and the final amino acids of the signal sequence have been altered. This allows restriction enzyme recognition sites EagI and Xbal to be present. Following gene iv is the phage origin of replication (ori). After ori is bla which confers resistance to ampicillin (ApR). The phage genes and bla are transcribed in the same sense.

After bla, is the Fab cassette (illustrated in FIG. 17) comprising: a) PlacZ promoter, b) A first Ribosome Binding Site (RBS1), c) The signal sequence form M13 iii, d) An ApaLI RERS, e) A light chain (a kappa L20::JK1 shortened by one codon at the V-J boundary in this case), f) An AscI RERS, g) A second Ribosome Binding Site (RBS2), h) A signal sequence, preferably PelB, which contains, i) An Sfil RERS, j) A synthetic 3-23 V region with diversity in CDR1 and CDR2, k) A captured CDR3, l) A partially synthetic J region (FR4 after BstEII), m) CHI, n) A NotI RERS, o) A His6 tag, p) A cMyc tag, q) An amber codon, r) An anchor DNA that encodes the same amino-acid sequence as codons 273 to 424 of M13 iii (as shown in Table 37). s) Two stop codons, t) An AvrII RERS, and u) A trp terminator.

The anchor (item r) encodes the same amino-acid sequence as do codons 273 to 424 of M13 iii but the DNA is approximately as different as possible from the wild-type DNA sequence. In Table 36, the III' stump runs from base 8997 to base 9455. Below the DNA, as comments, are the differences with wild-type iii for the comparable codons with "!W.T" at the ends of these lines. Note that Met and Trp have only a single codon and must be left as is. These AA types are rare. Ser codons can be changed at all three base, while Leu and Arg codons can be changed at two.

In most cases, one base change can be introduced per codon. This has three advantages: 1) recombination with the wild-type gene carried elsewhere on the phage is less likely, 2) new restriction sites can be introduced, facilitating construction; and 3) sequencing primers that bind in only one of the two regions can be designed.

The fragment of M13 III shown in CJRA05 is the preferred length for the anchor segment.

Alternative longer or shorter anchor segments defined by reference to whole mature III protein may also be utilized.

The sequence of M13 III consists of the following elements: Signal Sequence:: Domain 1 (D1) ::Linker 1 (Ll)::Domain 2 (D2)::Linker 2 (L2) :: Domain 3 (D3) :: Transmembrane Segment (TM) :: Intracellular anchor (IC) (see Table 38).

The pill anchor (also known as trpIII) preferably consists of D2::L2::D3::TM::IC. Another embodiment for the pill anchor consists of D2'::L2::D3::TM::IC (where D2' comprises the last 21 residues of D2 with the first 109 residues deleted). A further embodiment of the pill anchor consists of D2'(C>S)::L2::D3::TM::IC (where D2'(C>S) is D2’ with the single C converted to S), and d) D3::TM::IC.

Table 38 shows a gene fragment comprising the Not I site, His6 tag, cMyc tag, an amber codon, a recombinant enterokinase cleavage site, and the whole of mature M13 III protein. The DNA used to encode this sequence is intentionally very different from the DNA of wild-type gene iii as shown by the lines denoted "W.T." containing the w.t. bases where these differ from this gene. Ill is divided into domains denoted "domain 1", "linker 1", "domain 2", "linker 2", "domain 3", "transmembrane segment", and "intracellular anchor".

Alternative preferred anchor segments (defined by reference to the sequence of Table 38) include : codons 1-29 joined to codons 104-435, deleting domain 1 and retaining linker 1 to the end; codons 1-38 joined to codons 104-435, deleting domain land retaining the rEK cleavage site plus linker 1 to the end from III; codons 1-29 joined to codons 236-435, deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end; codons 1-38 joined to codons 236-435, deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site; codons 1-29 joined to codons 236-435 and changing codon 240 to Ser(e.g., age), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end; and codons 1-38 joined to codons 236-435 and changing codon 240 to Ser(e.g., age), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site.

The constructs would most readily be made by methods similar to those of Wang and Wilkinson (Biotechniaues 2001: 31(4)722-724) in which PCR is used to copy the vector except the part to be deleted and matching restriction sites are introduced or retained at either end of the part to be kept. Table 39 shows the oligonucleotides to be used in deleting parts of the III anchor segment. The DNA shown in Table 38 has an Nhel site before the DINDDRMA recombinant enterokinase cleavage site (rEKCS). If Nhel is used in the deletion process with this DNA, the rEKCS site would be lost. This site could be quite useful in cleaving Fabs from the phage and might facilitate capture of very high-afffinity antibodies. One could mutagenize this sequence so that the Nhel site would follow the rEKCS site, an Ala Ser amino-acid sequence is already present. Alternatively, one could use SphI for the deletions. This would involve a slight change in amino acid sequence but would be of no consequence.

Example 7 : Selection of antigen binders from an enriched library of human antibodies using phage vector DY3F31.

In this example the human antibody library used is described in de Haard et al., (Journal of Biological Chemistry. 274 (26): 18218-30 (1999). This library, consisting of a large non-immune human Fab phagemid library, was first enriched on antigen, either on streptavidin or on phenyl-oxazolone (phOx). The methods for this are well known in the art. Two preselected Fab libraries, the first one selected once on immobilized phOx-BSA (Rl-ox) and the second one selected twice on streptavidin (R2-strep), were chosen for recloning.

These enriched repertoires of phage antibodies, in which only a very low percentage have binding activity to the antigen used in selection, were confirmed by screening clones in an ELISA for antigen binding. The selected Fab genes were transferred from the phagemid vector of this library to the DY3F31 vector via ApaLl-Notl restriction sites. DNA from the DY3F31 phage vector was pretreated with ATP dependent DNAse to remove chromosomal DNA and then digested with ApaLl and Notl.

An extra digestion with AscI was performed in between to prevent self-ligation of the vector. The ApaLl/Notl Fab fragment from the preselected libraries was subsequently ligated to the vector DNA and transformed into competent XLl-blue MRF' cells.

Libraries were made using vector: insert ratios of 1:2 for phOx-library and 1:3 for STREP library, and using 100 ng ligated DNA per 50 μΐ of electroporation-competent cells (electroporation conditions : one shock of 1700 V, 1 hour recovery of cells in rich SOC medium, plating on amplicillin- containing agar plates).

This transformation resulted in a library size of 1.6 x 106 for Rl-ox in DY3F31 and 2.1 x 106 for R2-strep in DY3F31. Sixteen colonies from each library were screened for insert, and all showed the correct size insert (±1400 bp) (for both libraries).

Phage was prepared from these Fab libraries as follows. A representative sample of the library was inoculated in medium with ampicillin and glucose, and at OD 0.5, the medium exchanged for ampicillin and 1 mM IPTG. After overnight growth at 37 °C, phage was harvested from the supernatant by PEG-NaCl precipitation. Phage was used for selection on antigen. Rl-ox was selected on phOx-BSA coated by passive adsorption onto immunotubes and R2-strep on streptavidin coated paramagnetic beads (Dynal, Norway), in procedures described in de Haard et. al. and Marks et. al., Journal of Molecular Biology, 222(3): 581-97 (1991). Phage titers and enrichments are given in Table 40.

Clones from these selected libraries, dubbed R2-ox and R3-strep respectively, were screened for binding to their antigens in ELISA. 44 clones from each selection were picked randomly and screened as phage or soluble Fab for binding in ELISA. For the libraries in DY3F31, clones were first grown in 2TY-2% glucose-50 pg/ml AMP to an OD600 of approximately 0.5, and then grown overnight in 2TY-50 pg/ml AMP + /- ImM IPTG. Induction with IPTG may result in the production of both phage-Fab and soluble Fab. Therefore the (same) clones were also grown without IPTG. Table 41 shows the results of an ELISA screening of the resulting supernatant, either for the detection of phage particles with antigen binding (Anti-M13 HRP = anti-phage antibody), or for the detection of human Fabs, be it on phage or as soluble fragments, either with using the anti-myc antibody 9E10 which detects the myc-tag that every Fab carries at the C-terminal end of the heavy chain followed by a HRP-labeled rabbit-anti-Mouse serum (column 9E10/RAM-HRP), or with anti-light chain reagent followed by a HRP-labeled goat-anti-rabbit antiserum(anti-CK/CL Gar-HRP) .

The results shows that in both cases antigen-binders are identified in the library, with as Fabs on phage or with the anti-Fab reagents (Table 41). IPTG induction yields an increase in the number of positives. Also it can be seen that for the phOx-clones, the phage ELISA yields more positives than the soluble Fab ELISA, most likely due to the avid binding of phage. Twenty four of the ELISA-positive clones were screened using PCR of the Fab-insert from the vector, followed by digestion with BstNI. This yielded 17 different patterns for the phOx-binding

Fab's in 23 samples that were correctly analyzed, and 6 out of 24 for the streptavidin binding clones. Thus, the data from the selection and screening from this pre-enriched non-immune Fab library show that the DY3F31 vector is suitable for display and selection of Fab fragments, and provides both soluble Fab and Fab on phage for screening experiments after selection.

Example 8: Selection of Phage-antibody libraries on streptavidin magnetic beads .

The following example describes a selection in which one first depletes a sample of the library of binders to streptavidin and optionally of binders to a non-target (i.e., a molecule other than the target that one does not want the selected Fab to bind). It is hypothesized that one has a molecule, termed a "competitive ligand", which binds the target and that an antibody which binds at the same site would be especially useful.

For this procedure Streptavidin Magnetic Beads (Dynal) were blocked once with blocking solution (2% Marvel Milk, PBS (pH 7.4), 0.01% Tween-20 ("2%MPBST")) for 60 minutes at room temperature and then washed five times with 2%MPBST. 450 pL of beads were blocked for each depletion and subsequent selection set.

Per selection, 6.25 pL of biotinylated depletion target (1 mg/mL stock in PBST) was added to 0.250 mL of washed, blocked beads (from step 1). The target was allowed to bind overnight, with tumbling, at 4°C. The next day, the beads are washed 5 times with PBST.

Per selection, 0.010 mL of biotinylated target antigen (1 mg/mL stock in PBST) was added to 0.100 mL of blocked and washed beads (from step 1).

The antigen was allowed to bind overnight, with tumbling, at 4°C. The next day, the beads were washed 5 times with PBST.

In round 1, 2 X 1012 up to 1013 plaque forming units (pfu) per selection were blocked against non-specific binding by adding to 0.500 mL of 2%MPBS (=2%MPBST without Tween) for 1 hr at RT (tumble). In later rounds, 1011 pfu per selection were blocked as done in round 1.

Each phage pool was incubated with 50 pL of depletion target beads (final wash supernatant removed just before use) on a Labquake rotator for 10 min at room temperature. After incubation, the phage supernatant was removed and incubated with another 50 pL of depletion target beads. This was repeated 3 more times using depletion target beads and twice using blocked streptavidin beads for a total of 7 rounds of depletion, so each phage pool required 350 pL of depletion beads. A small sample of each depleted library pool was taken for titering. Each library pool was added to 0.100 mL of target beads (final wash supernatant was removed just before use) and allowed to incubate for 2 hours at room temperature (tumble).

Beads were then washed as rapidly as possible (e.g.,3 minutes total) with 5 X 0.500 mL PBST and then 2X with PBS. Phage still bound to beads after the washing were eluted once with 0.250 mL of competitive ligand (-1 ppM) in PBST for 1 hour at room temperature on a Labquake rotator. The eluate was removed, mixed with 0.500 mL Minimal A salts solution and saved. For a second selection, 0.500 mL 100 mM TEA was used for elution for 10 min at RT, then neutralized in a mix of 0.250 mL of 1 M Tris, pH 7.4 + 0.500 mL Min A salts.

After the first selection elution, the beads can be eluted again with 0.300 mL of non-biotinylated target (1 mg/mL) for 1 hr at RT on a Labquake rotator. Eluted phage are added to 0.450 mL Minimal A salts.

Three eluates (competitor from 1st selection, target from 1st selection and neutralized TEA elution from 2nd selection) were kept separate and a small aliquot taken from each for titering. 0.500 mL Minimal A salts were added to the remaining bead aliquots after competitor and target elution and after TEA elution. Take a small aliquot from each was taken for tittering.

Each elution and each set of eluted beads was mixed with 2X YT and an aliquot (e.g., 1 mL with 1. E 10/mL) of XLl-Blue MRF' E. coli cells (or other F' cell line) which had been chilled on ice after having been grown to mid-logarithmic phase, starved and concentrated (see procedure below - "Mid-Log prep of XL-1 blue MRF' cells for infection").

After approximately 30 minutes at room temperature, the phage/cell mixtures were spread onto Bio-Assay Dishes (243 X 243 X 18 mm, Nalge Nunc) containing 2XYT, ImM IPTG agar. The plates were incubated overnight at 30°C. The next day, each amplified phage culture was harvested from its ' respective plate. The plate was flooded with 35 mL TBS or LB, and cells were scraped from the plate. The resuspended cells were transferred to a centrifuge bottle. An additional 20 mL TBS or LB was used to remove any cells from the plate and pooled with the cells in the centrifuge bottle. The cells were centrifuged out, and phage in the supernatant was recovered by PEG precipitation. Over the next day, the amplified phage preps were titered.

In the first round, two selections yielded five amplified eluates. These amplified eluates were panned for 2-3 more additional rounds of selection using ~1. E 12 input phage/round. For each additional round, the depletion and target beads were prepared the night before the round was initiated.

For the elution steps in subsequent rounds, all elutions up to the elution step from which the amplified elution came from were done, and the previous elutions were treated as washes. For the bead infection amplified·phage, for example, the competitive ligand and target elutions were done and then tossed as washes (see below). Then the beads were used to infect E. coli. Two pools, therefore, yielded a total of 5 final elutions at the end of the selection. 1st selection set A. Ligand amplified elution: elute w/ ligand for 1 hr, keep as elution B. Target amplified elution: elute w/ ligand for 1 hr, toss as wash elute w/ target for 1 hr, keep as elution C. Bead infect, amp. elution: elute w/ ligand for 1 hr, toss as wash elute w/ target for 1 hr, toss as wash elute w/ cell infection, keep as elution 2nd selection set A. TEA amplified elution; elute w/ TEA lOmin, keep as elution B. Bead infect, amp. elution; elute w/ TEA lOmin, toss as wash elute w/ cell infection, keep as elution

Mid-log prep of XLl blue MRF' cells for infection (based on Barbas et al. Phage Display manual procedure)

Culture XLl blue MRF' in NZCYM (12.5 mg/mL tet) at 37°C and 250 rpm overnight. Started a 500 mL culture in 2 liter flask by diluting cells 1/50 in NZCYM/tet (10 mL overnight culture added) and incubated at 37°C at 250 rpm until OD600 of 0.45 (1.5-2 hrs) was . reached. Shaking was reduced to 100 rpm for 10 min.

When OD600 reached between 0.55-0.65, cells were transferred to 2 x 250 mL centrifuge bottles, centrifuged at 600 g for 15 min at 4°C. Supernatant was poured off. Residual liguid was removed with a pipette.

The pellets were gently resuspended (not pipetting up and down) in the original volume of 1 X Minimal A salts at room temp. The resuspended cells were transferred back into 2-liter flask, shaken at 100 rpm for 45 min at 37°C. This process was performed in order to starve the cells and restore pili. The cells were transferred to 2 x 250 mL centrifuge bottles, and centrifuged as earlier.

The cells were gently resuspended in ice cold Minimal A salts (5 mL per 500 mL original culture).

The cells were put on ice for use in infections as soon as possible.

The phage eluates were brought up to 7.5 mL with 2XYT medium and 2.5 mL of cells were added. Beads were brought up to 3 mL with 2XYT and 1 mL of cells were added. Incubated at 37oC for 30 min. The cells were plated on 2XYT, 1 mM IPTG agar large NUNC plates and incubated for 18 hr at 30°C.

Example 9: Incorporation of synthetic region in FR1/3 region.

Described below are examples for incorporating of fixed residues in antibody sequences for light chain kappa and lambda genes, and for heavy chains. The experimental conditions and oligonucleotides used for the examples below have been described in previous examples (e.g., Examples 3 &amp; 4).

The process for incorporating fixed FRl residues in an antibody lambda sequence consists of 3 steps (see FIG. 18): (1) annealing of single-stranded DNA material encoding VL genes to a partially complementary oligonucleotide mix (indicated with Ext and Bridge), to anneal in this example to the region encoding residues 5-7 of the FRl of the lambda genes (indicated with X..X; within the lambda genes the overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VL gene. In this process the first few residues of all lambda genes will be encoded by the sequences present in the oligonucleotides (Ext., Bridge or PCRpr). After the PCR, the lambda genes can be cloned using the indicated restriction site for ApaLI.

The process for incorporating fixed FRl residues in an antibody kappa sequence (FIG. 19) consists of 3 steps : (1) annealing of single-stranded DNA material encoding VK genes to a partially complementary oligonucleotide mix (indicated with Ext and Bri), to anneal in this example to the region encoding residues 8-10 of the FRl of the kappa genes (indicated with X..X; within the kappa genes the overlap may sometimes not be perfect) ; (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VK gene. In this process the first few (8) residues of all kappa genes will be encode by the sequences present in the oligonucleotides (Ext., Bridge or PCRpr.). After the PCR, the kappa genes can be cloned using the indicated restriction site for ApaLI.

The process of incorporating fixed FR3 residues in a antibody heavy chain sequence (FIG. 20) consists of 3 steps : (1) annealing of single-stranded DNA material encoding part of the VH genes (for example encoding FR3, CDR3 and FR4 regions) to a partially complementary oligonucleotide mix (indicated with Ext and Bridge), to anneal in this example to the region encoding residues 92-94 (within the FR3 region) of VH genes (indicated with X..X; within the VH genes the overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VH gene (such as in the FR4 region).

In this process certain residues of all VH genes will be encoded by the sequences present in the oligonucleotides used here, in particular from PCRpr (for residues 70-73), or from Ext/Bridge oligonucleotides (residues 74-91). After the PCR, the partial VH genes can be cloned using the indicated restriction site for Xbal.

It will be understood that the foregoing is only illustrative of the principles of this invention and that various modifications can be made by those skilled in the art without departing from the scope of and sprit of the invention.

The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Table 1: Human GLG FR3 sequences ! VH1 ! 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 agg gtc acc atg acc agg gac acg tcc ate age aca gcc tac atg ! 81 82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92 gag ctg age agg ctg aga tet gac gac acg gcc gtg tat tac tgt ! 93 94 95 geg aga ga ! 1-02# 1 aga gtc acc att acc agg gac aca tec geg age aca gcc tac atg gag ctg age age ctg aga tet gaa gac acg get gtg tat tac tgt geg aga ga ! 1-03# 2 aga gtc acc atg acc agg aac acc tec ata age aca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt geg aga gg ! 1-08# 3 aga gtc acc atg acc aca gac aca tec acg age aca gcc tac atg gag ctg agg age ctg aga tet gac gac acg gcc gtg tat tac tgt geg aga ga ! 1-18# 4 aga gtc acc atg acc gag gac aca tet aca gac aca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt gca aca ga ! 1-24# 5 aga gtc acc att acc agg gac agg tet atg age aca gcc tac atg gag ctg age age ctg aga tet gag gac aca gcc atg tat tac tgt gca aga ta ! 1-45# 6 aga gtc acc atg acc agg gac acg tec acg age aca gtc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt geg aga ga ! 1-46# 7 aga gtc acc att acc agg gac atg tec aca age aca gcc tac atg gag ctg age age ctg aga tec gag gac acg gcc gtg tat tac tgt geg gca ga ! 1-58# 8 aga gtc acg att acc geg gac gaa tec acg age aca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt geg aga ga ! 1-69# 9 aga gtc acg att acc geg gac aaa tec acg age aca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt geg aga ga ! 1-e# 10 aga gtc acc ata acc geg gac acg tet aca gac aca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg tat tac tgt gca aca ga ! 1-f# 11 ! VH2 agg etc acc ate acc aag gac ace tee aaa aac cag gtg gtc ett aca atg acc aac atg gac cct gtg gac aca gee aca tat tac tgt gca cac aga c! 2-05# 12 agg etc acc ate tee aag gac acc tee aaa age cag gtg gtc ett acc atg acc aac atg gac cct gtg gac aca gee aca tat tac tgt gca egg ata c! 2-26# 13 agg etc acc ate tee aag gac acc tee aaa aac cag gtg gtc ett aca atg acc aac atg gac cct gtg gac aca gee aeg tat tac tgt gca egg ata c! 2-70# 14 ! VH3 ega ttc acc ate tee aga gac aac gee aag aac tea ctg tat ctg caa atg aac age ctg aga gee gag gac aeg get gtg tat tac tgt geg aga ga ! 3-07# 15 ega ttc acc ate tee aga gac aac gee aag aac tee ctg tat ctg caa atg aac agt ctg aga get gag gac aeg gcc.ttg tat tac tgt gca aaa gat a! 3-09116 ega ttc acc ate tee agg gac aac gee aag aac tea ctg tat ctg caa atg aac age ctg aga gee gag gac aeg gee gtg tat tac tgt geg aga ga ! 3-11# 17 ega ttc acc ate tee aga gaa aat gee aag aac tee ttg tat ett caa atg aac age ctg aga gee ggg gac aeg get gtg tat tac tgt gca aga ga ! 3-13# 18 . aga ttc acc ate tea aga gat gat tea aaa aac aeg ctg tat ctg caa atg aac age ctg aaa acc gag gac aca gee gtg tat tac tgt acc aca ga ! 3-15# 19 ega ttc acc ate tee aga gac aac gee aag aac tee ctg tat ctg caa atg aac agt ctg aga gee gag gac aeg gee ttg tat cac tgt geg aga ga ! 3-20# 20 ega ttc acc ate tee aga gac aac gee aag aac tea ctg tat ctg caa atg aac age ctg aga gee gag gac aeg get gtg tat tac tgt geg aga ga ! 3-21# 21 egg ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ctg caa atg aac age ctg aga gee gag gac aeg gee gta tat tac tgt geg aaa ga ! 3-23# 22 ega ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ctg caa atg aac age ctg aga get gag gac aeg get gtg tat tac tgt geg aaa ga ! 3-30# 23 ega ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ctg caa atg aac age ctg aga get gag gac aeg get gtg tat tac tgt geg aga ga ! 3303# 24 cga ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ctg caa atg aac age ctg aga get gag gac aeg get gtg tat tac tgt geg aaa ga ! 3305# 25 cga ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ctg caa atg aac age ctg aga gee gag gac aeg get gtg tat tac tgt geg aga ga ! 3-33# 26 cga ttc acc ate tee aga gac aac age aaa aac tee ctg tat ctg caa atg aac agt ctg aga act gag gac acc gee ttg tat tac tgt gca aaa gat a! 3-43#27 cga ttc acc ate tee aga gac aat gee aag aac tea ctg tat ctg caa atg aac age ctg aga gac gag gac aeg get gtg tat tac tgt geg aga ga ! 3-48# 28 aga ttc acc ate tea aga gat ggt tee aaa age ate gee tat ctg caa atg aac age ctg aaa acc gag gac aca gee gtg tat tac tgt act aga ga ! 3-49# 29 cga ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ett caa atg aac age ctg aga gee gag gac aeg gee gtg tat tac tgt geg aga ga ! 3-53# 30 aga ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ett caa atg ggc age ctg aga get gag gac atg get gtg tat tac tgt geg aga ga ! 3-64# 31 aga ttc acc ate tee aga gac aat tee aag aac aeg ctg tat ett caa atg aac age ctg aga get gag gac aeg get gtg tat tac tgt geg aga ga ! 3-66# 32 aga ttc acc ate tea aga gat gat tea aag aac tea ctg tat ctg caa atg aac age ctg aaa acc gag gac aeg gee gtg tat tac tgt get aga ga ! 3-72# 33 agg ttc acc ate tee aga gat gat tea aag aac aeg geg tat ctg caa atg aac age ctg aaa acc gag gac aeg gee gtg tat tac tgt act aga ca ! 3-73# 34 cga ttc acc ate tee aga gac aac gee aag aac aeg ctg tat ctg caa atg aac agt ctg aga gee gag gac aeg get gtg tat tac tgt gca aga ga ! 3-74# 35 aga ttc acc ate tee aga gac aat tee aag aac aeg ctg cat ett caa atg aac age ctg aga get gag gac aeg get gtg tat tac tgt aag aaa ga ! 3-d# 36 ! VH4 cga gtc acc ata tea gta gac aag tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee geg gac aeg gee gtg tat tac tgt geg aga ga ! 4-04# 37 cga gtc acc atg tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee gtg gac aeg gee gtg tat tac tgt geg aga aa ! 4-28# 38 ega gtt acc ata tea gta gac aeg tet aag aac cag ttc tee ctg aag ctg age tet gtg act gee geg gac aeg gee gtg tat tac tgt geg aga ga ! 4301# 39 ega gtc acc ata tea gta gac agg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee geg gac aeg gee gtg tat tac tgt gee aga ga ! 4302# 40 ega gtt acc ata tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg act gee gca gac aeg gee gtg tat tac tgt gee aga ga ! 4304# 41 ega gtt acc ata tea gta gac aeg tet aag aac cag ttc tee ctg aag ctg age tet gtg act gee geg gac aeg gee gtg tat tac tgt geg aga ga ! 4-31# 42 ega gtc acc ata tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee geg gac aeg get gtg tat tac tgt geg aga ga ! 4-34# 43 ega gtc acc ata tee gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee gca gac aeg get gtg tat tac tgt geg aga ca ! 4-39# 44 ega gtc acc ata tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc get geg gac aeg gee gtg tat tac tgt geg aga ga ! 4-59# 45 ega gtc acc ata tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc get geg gac aeg gee gtg tat tac tgt geg aga ga ! 4-61# 46 ega gtc acc ata tea gta gac aeg tee aag aac cag ttc tee ctg aag ctg age tet gtg acc gee gca gac aeg gee gtg tat tac tgt geg aga ga ! 4-b# 47 ! VH5 cag gtc acc ate tea gee gac aag tee ate age acc gee tac ctg cag tgg age age ctg aag gee teg gac acc gee atg tat tac tgt geg aga ca ! 5-51# 48 cac gtc acc ate tea get gac aag tee ate age act gee tac ctg cag tgg age age ctg aag gee teg gac acc gee atg tat tac tgt geg aga ! 5-a# 49 ! VH6 ega ata acc ate aac cca gac aca tee aag aac cag ttc tee ctg cag ctg aac tet gtg act ccc gag gac aeg get gtg tat tac tgt gca aga ga ! 6-1# 50 ! VH7 egg ttt gtc ttc tee ttg gac acc tet gtc age aeg gca tat ctg cag ate tgc age eta aag get gag gac act gee gtg tat tac tgt geg aga ga ! 74.1# 51

Table 2: Enzymes that either cut 15 or more human GLGs or have 5+-base recognition in FR3

Typical entry: REname Recognition #sites GLGid#:base# GLGid#:base# GLGid#:base#.....

BstEII Ggtnacc 2 L: 3 48: 3

There are 2 hits at base# 3

Maelll gtnac 36 1: 4 2: 4 3: 4 4: 4 5: 4 6: 4 7: 4 8: 4 9: 4 10: 4 11: 4 37: 4 37: 58 38: 4 38: 58 39: 4 39: 58 40: 4 40: 58 41: 4 41: 58 42: 4 42: 58 43: 4 43: 58 44: 4 44: 58 45: 4 45: 58 46: 4 46: 58 47: 4 47: 58 48: 4 49: 4 50: 58

There are 24 hits at base# 4

Tsp45I gtsac 33 1: 4 2: 4 3: 4 4: 4 5: 4 6: 4 7: 4 8: 4 9: 4 10: 4 11: 4 37: 4 37: 58 38: 4 38: 58 39: 58 40: 4 40: 58 41: 58 42: 58 43: 4 43: 58 44: 4 44: 58 45: 4 45: 58 46: 4 46: 58 47: 4 47: 58 48: 4 49: 4 50: 58 ‘

There are 21 hits at base# 4

HphI tcacc 45 1: 5 2: 5 3: 5 4: 5 5: 5 6: 5 7: 5 8: 5 11: 5 12: 5 12: 11 13: 5 14: 5 15: 5 16: 5 17: 5 18: 5 19: 5 20: 5 21: 5 22: 5 23: 5 24: 5 25: 5 26: 5 27: 5 28: 5 29: 5 30: 5 31: 5 32: 5 33: 5 34: 5 35: 5 36: 5 37: 5 ' 38: 5 40: 5 43: 5 44: 5 45: 5 46: 5 47: 5 48: 5 49: 5

There are 44 hits at base# 5

Nlalll CATG 26 1: 9 1: 42 2: 42 3: 9 3: 42 4: 9 4: 42 5: 9 5: 42 6: 42 6: 78 7: 9 7: 42 8: 21 8: 42 9: 42 10: 42 11: 42 12: 57 13: 48 13: 57 14: 57 31: 72 38: 9 48: 78 49: 78

There are 11 hits at base# 42

There are 1 hits at base# 48 Could cause raggedness.

BsaJI Ccnngg 37 1: 14 2: 14 5: 14 6: 14 7: 14 8: 14 8: 65 9: 14 10: 14 11: 14 12: 14 13: 14 14: 14 15: 65 17: 14 17: 65 18: 65 19: 65 20: 65 21: 65 22: 65 26: 65 29: 65 30: 65 33: 65 34: 65 35: 65 37: 65 38: 65 39: 65 40: 65 42: 65 43: 65 48: 65 49: 65 50: 65 51: 14

There are 23 hits at base# 65

There are 14 hits at base# 14

Alul AGct ' 42 1: 47 2: 47 3: 47 4: 47 5: 47 6: 47 7: 47 8: 47 9: 47 10: 47 11: 47 16: 63 23: 63 24: 63 25: 63 31: 63 32: 63 36: 63 37: 47 37: 52 38: 47 38: 52 39: 47 39: 52 40: 47 40: 52 41: 47 41: 52 42: 47 42: 52 43: 47 43: 52 44: 47 44: 52 45: 47 45: 52 46: 47 46: 52 47: 47 47: 52 49: 15 50: 47

There are 23 hits at base# 47

There are 11 hits at base# 52 Only 5 bases from 47

BlpI GCtnagc 21 1: 48 2: 48 3: 48 5: 48 6: 48 7: 48 8: 48 9: 48 10: 48 11: 48 37: 48 38: 48 39: 48 40: 48 41: 48 42: 48 43: 48 44: 48 45: 48 46: 48 47: 48

There are 21 hits at base# 48

Mwol GCNNNNNnngc 19 1: 48 2: 28 19: 36 22: 36 23: 36 24: 36 25: 36 26: 36 35: 36 37: 67 39: 67 40: 67 41: 67 42: 67 43: 67 44: 67 45: 67 46: 67 47: 67

There are 10 hits at base# 67

There are 7 hits at base# 36

Ddel Ctnag 71 1: 49 1: 58 2: 49 2: 58 3: 49 3: 58 3: 65 4: 49 4: 58 5: 49 5: 58 5: 65 6: 49 6: 58 6: 65 7: 49 7: 58 7: 65 8: 49 8: 58 9: 49 9: 58 9: 65 10: 49 10: 58 10: 65 11: 49 11: 58 11: 65 15: 58 16: 58 16: 65 17: 58 18: 58 20: 58 21: 58 22: 58 23: 58 23: 65 24: 58 24: 65 25: 58 25: 65 26: 58 27: 58 27: 65 28: 58 30: 58 31: 58 31: 65 32: 58 32: 65 35: 58 36: 58 36: 65 37: 49 38: 49 39: 26 39: 49 40: 49 41: 49 42: 26 42: 49 43: 49 44: 49 45: 49 46: 49 47: 49 48: 12 49: 12 51: 65

There are 29 hits at base# 58

There are 22 hits at base# 49 Only nine base from 58

There are 16 hits at base# 65 Only seven bases from 58

Bglll Agatct 11 1: 61 2: 61 3: 61 4: 61 5: 61 6: 61 7: 61 9: 61 10: 61 11: 61 51: 47

There are 10 hits at base# 61

BstYI Rgatcy 12 1: 61 2: 61 3: 61 4: 61 5: 61 6: 61 7: 61 8: 61 9: 61 10: 61 11: 61 51: 47

There are 11 hits at base# 61

Hpyl88I TCNga 17 1: 64 2: 64 3: 64 4: 64 5: 64 6: 64 7: 64 8: 64 9: 64 10: 64 11: 64 16: 57 20: 57 27: 57 35: 57 48: 67 49: 67

There are 11 hits at base# 64

There are 4 hits at base# 57

There are 2 hits at base# 67 Could be ragged.

MslI CAYNNnnRTG 44 1: 72 2: 72 3: 72 4: 72 5: 72 6: 72 7: 72 8: 72 9: 72 10: 72 11: 72 15: 72 17: 72 18: 72 19: 72 21: 72 23: 72 24: 72 25: 72 26: 72 28: 72 29: 72 30: 72 31: 72 32: 72 33: 72 34: 72 35: 72 36: 72 37: 72 38: 72 39: 72 40: 72 41: 72 42: 72 43: 72 44: 72 45: 72 46: 72 47: 72 48: 72 49: 72 50: 72 51: 72

There are 44 hits at base# 72

BsiEI CGRYcg 23 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74

There are 23 hits at base# 74

Eael Yggccr 23 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74

There are 23 hits at base# 74

EagI Cggecg 23 1: 74 3: 74 4: 74 5: 74 7: 74 8: 74 9: 74 10: 74 11: 74 17: 74 22: 74 30: 74 33: 74 34: 74 37: 74 38: 74 39: 74 40: 74 41: 74 42: 74 45: 74 46: 74 47: 74

There are 23 hits at base# 74

Haelll GGcc 27 1: 75 3: 75 4: 75 5: 75 7: 75 8: 75 9: 75 10: 75 11: 75 16: 75 17: 75 20: 75 22: 75 30: 75 33: 75 34: 75 37: 75 38: 75 39: 75 40: 75 41: 75 42: 75 45: 75 46: 75 47: 75 48: 63 49: 63

There are 25 hits at base# 75

Bst4CI ACNgt 65°C 63 Sites There is a third isoschismer 1: 86 2: 86 3: 86 4: 86 5: 86 6: 86 7: 34 7: 86 8: 86 9: 86 10: 86 11: 86 12: 86 13: 86 14: 86 15: 36 15: 86 16: 53 16: 86 17: 36 17: 86 18: 86 19: 86 20: 53 20: 86 21: 36 21: 86 22: 0 22: 86 23: 86 24: 86 25: 86 26: 86 27: 53 27: 86 28: 36 28: 86 29: 86 30: 86 31: 86 32: 86 33: 36 33: 86 34: 86 35: 53 35: 86 36: 86 37: 86 38: 86 39: 86 40: 86 41: 86 42: 86 43: 86 44: 86 45: 86 46: 86 47: 86 48: 86 49: 86 50: 86 51: 0 51: 86

There are 51 hits at base# 86 All the other sites are well away

HpyCH4III ACNgt 63 1: 86 2: 86 3: 86 4: 86 5: 86 6: 86 7: 34 7: 86 8: 86 9: 86 10: 86 11: 86 12: 86 13: 86 14: 86 15: 36 15: 86 16: 53 16: 86 17: 36 17: 86 18: 86 19: 86 20: 53 20: 86 21: 36 21: 86 22: 0 22: 86 23: 86 24: 86 25: 86 26: 86 27: 53 27: 86 28: 36 28: 86 29: 86 30: 86 31: 86 32: 86 33: 36 33: 86 34: 86 35: 53 35: 86 36: 86 37: 86 38: 86 39: 86 40: 86 41: 86 42: 86 43: 86 44: 86 45: 86 46: 86 47: 86 48: 86 49: 86 50: 86 51: 0 51: 86

There are 51 hits at base# 86

Hinfl Gantc 43 2:2 3:2 4:2 5:2 6:2 7:2 B: 2 9: 2 9: 22 10: 2 11: 2 15: 2 16: 2 17: 2 18: 2 19: 2 19: 22 20: 2 21: 2 23: 2 24: 2 25: 2 26: 2 27: 2 28: 2 29: 2 30: 2 31: 2 32: 2 33: 2 33: 22 34: 22 35: 2 36: 2 37: 2 38: 2 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2 50: 60

There are 38 hits at base# 2

Mlyl GAGTCNNNNNn 18 2;2 3:2 4:2 5:2 6:2 7:2 g: 2 9: 2 10: 2 11: 2 37: 2 38: 2 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2

There are 18 hits at base# 2

Plel gagtc 18 2;2 3:2 4:2 5:2 6:2 7:2 8: 2 9: 2 10: 2 11: 2 37: 2 38: 2 40: 2 43: 2 44: 2 45: 2 46: 2 47: 2

There are 18 hits at base# 2

Acil Ccgc 24 2; 26 9: 14 10: 14 11: 14 27: 74 37: ,_62— 37: 65 38: 62 39: 65 40: 62 40: 65 41: 65 42: 65 43: 62 43: 65 44: 62 44: 65 45: 62 46: 62 47: 62 47: 65 48: 35 48: 74 49: 74

There are 8 hits at base# 62

There are 8 hits at base# 65

There are 3 hits at base# 14

There are 3 hits at base# 74

There are 1 hits at base# 26

There are 1 hits at base# 35

Gcgg 11 8: 91 9: 16 10: 16 11: 16 37: 67 39: 67 40: 67 42: 67 43: 67 45: 67 46: 67

There are 7 hits at base# 67

There are 3 hits at base# 16

There are 1 hits at base# 91

BsiHKAI GWGCWc 20 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51 '

There are 11 hits at base# 51

Bspl28 61 GDGCHc 20 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51

There are 11 hits at base# 51

HgiAI GWGCWc 20 2: 30 4: 30 6: 30 7: 30 9: 30 10: 30 12: 89 13: 89 14: 89 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51

There are 11 hits at base# 51 1

BsoFI GCngc 26 2: 53 3: 53 5: 53 6: 53 7: 53 8: 53 8: 91 9: 53 10: 53 11: 53 31: 53 36: 36 37: 64 39: 64 40: 64 41: 64 42: 64 43: 64 44: 64 45: 64 46: 64 47: 64 48: 53 49: 53 50: 45 51: 53

There are 13 hits at base# 53 There are 10 hits at base# 64

Tsel Gcwgc 1^ 2: 53 3: 53 5: 53 6: 53 7: 53 8: 53 9: 53 10: 53 11: 53 31: 53 36: 36 45: 64 46: 64 48: 53 49: 53 50: 45 51: 53

There are 13 hits at base# 53 34

Mnll gagg 3: 67 3: 95 4: 51 5: 16 5: 67 6: 67 7: 67 8: 67 9: 67 10: 67 11: 67 15: 67 16: 67 17: 67 19: 67 20: 67 21: 67 22: 67 23: 67 24: 67 25: 67 26: 67 27: 67 28: 67 29: 67 -30: 67 31: 67 32: 67 33: 67 34: 67 35: 67 36: 67 50: 67 51: 67

There are 31 hits at base# 67

HpyCH4V TGca 34 5: 90 6: 90 11: 90 12: 90 13: 90 14: 90 15: 44 16: 44 16: 90 17: 44 18: 90 19: 44 20: 44 21: 44 22: 44 23: 44 24: 44 25: 44 26: 44 27: 44 27: 90 28: 44 29: 44 33: 44 34: 44 35: 44 35: 90 36: 38 48: 44 49: 44 50: 44 50: 90 51: 44 51: 52

There are 21 hits at base# 44 are 1 hits at base# 52 „ , 13 5-base recognition

AccI GTmkac 7: 37 11: 24 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 43: 16 44: 16 45: 16 46: 16 47: 16

There are 11 hits at base# 16 S.CII CCGCgg « 6'b“e ™<=°9nition 9: 14 10: 11 ll! 14 31: «5 39: 65 40: 65 42: 65 43: 65

There are 5 hits at base# 65 i There are 3 hits at base# 14

Tfil Gawtc 24 9; 22 15: 2 16: 2 17: 2 18: 2 19: 2 19: 22 20: 2 21: 2 23: 2 24: 2 25: 2 3 26: 2 27: 2 28: 2 29: 2 30: 2 31: 2 32: 2 33: 2 33: 22 34: 22 35: 2 36: 2 are 20 hits at base# 2

BsraAI Nnnnnngagac 19 15: 11 16: 11 20: 11 21: 11 22: 11 23: 11 24: 11 25: 11 26: 11 27: 11 28: 11 28: 56 30: 11 31: 11 32: 11 35: 11 36: 11 44: 87 48: 87

There are 16 hits at base# 11

Bpml ctccag 19 15: 12 16: 12 17: 12 18: 12 20: 12 21: 12 22: 12 23: 12 24: 12 25: 12 26: 12 27: 12 28: 12 30: 12 31: 12 32: 12 34: 12 35: 12 36: 12

There are 19 hits at base# 12

XmnI GAANNnnttc 12 37: 30 38: 30 39: 30 40: 30 41: 30 42: 30 43: 30 44: 30 45: 30 46: 30 47: 30 50: 30

There are 12 hits at base# 30

BsrI NCcagt 12 37: 32 38: 32 39: 32 40: 32 41: 32 42: 32 43: 32 44: 32 45: 32 46: 32 47: 32 50: 32

There are 12 hits at base# 32

Banll GRGCYc H 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51

There are 11 hits at base# 51

EC1136I GAGctc H 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51

There are 11 hits at base# 51

SacI GAGCTc 11 37: 51 38: 51 39: 51 40: 51 41: 51 42: 51 43: 51 44: 51 45: 51 46: 51 47: 51

There are 11 hits at base# 51

Table 3: Synthetic 3-23 FR3 of human heavy chains showning positions of possible cleavage sites

! Sites engineered into the synthetic gene are shown in upper case DNA ! with the RE name between vertical bars (as in | Xbal I ) . ! RERSs frequently found in GLGs are shown below the synthetic sequence ! with the name to the right (as in gtn ac=MaeIII(24), indicating that ! 24 of the 51 GLGs contain the site) .

I , |---FR3--- , 89 90 (codon # in

i R F synthetic 3-23) [cgc|ttc| 6 ! Allowed DNA |cgn|tty| I lagrl i ga ntc =

Hinfl(38) , ga gtc =

Plel(18) i ga wtc =

Tfil(20) i gtn ac =

Maelll(24) i gts ac =

Tsp45I(21) I tc acc =

HphI(44)

I I --------FR3-------------------------------------------------- i 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! TISRDNSKNTLYLQM I act I ate ITCTIAGA1gacIaacItetIaagIaatI act|etc (tacIttgIcagIatg| 51 !allowed|acnIath|ten Icgn|gay IaayI ten IaarIaayIacnIttrItayIttrI car Iatg| ! |agy|agr| lagyl Ictn| Ictnl 1 | galgac = BsmAI(16) ag ct =

Alul(23) ! cItcc ag = Bpml(19) 9 ctn age =

BlpI(21) ! ιι g aan nnn ttc = Xmnl(12) I | xbaI | tg ca = HpyCH4V(21) 1 ! ------------------------------------------------------------- ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! NSLRAEDTAVYYCAK

IaacIagCITTA|AGgI get I gag IgacIaCT|GCA | GtcItacI tat ItgcI get IaaaI 96 !allowed IaayI ten Ittr|cgnI gen I gar I gay IacnI gen IgtnItayItayItgyI gen|aarI ! IagyIctnIagrI I I I | | cc nng g = BsaJI(23) ac ngt = Bst4CI(51> ! | aga tet = Bglll(lO) I ac ngt = HpyCH4III(51) ! | Rga tcY = BstYI(ll) I ac ngt = Taal<51) I || c ayn nnn rtc = Mali(44) I || eg rye g = BsiEI(23) ! || yg gee r = Eael(23) I || eg gee g = EagI(23) ! || |g gee = Haelll(25) ! | | gag g = Mnll(31)1

! |Aflll I I PstI I

Table 4: REdaptors, Extenders, and Bridges used for Cleavage and Capture of Human Heavy Chains in FR3. A: HpyCH4V Probes of actual human HC genes !HpyCH4V in FR3 of human HC, bases 35-56; only those with TGca site TGca;10, RE recognition:tgca of length 4 is expected at 10 1 6-1 agttctccctgcagctgaactc 2 3-11, 3-07,3-21,3-72,3-48 cactgtatctgcaaatgaacag 3 3-09,3-43,3-20 ccctgtatctgcaaatgaacag 4 5-51 ccgcctacctgcagtggagcag 5 3-15,3-30,3-30.5,3-30.3,3-74,3-23,3-33 cgctgtatctgcaaatgaacag 6 7-4.1 cggcatatctgcagatctgcag 7 3-73 cggcgtatctgcaaatgaacag g 5-a ctgcctacctgcagtggagcag 9 3-49 tcgcctatctgcaaatgaacag B: HpyCH4V REdaptors, Extenders, and Bridges B.l REdaptors ! Cutting HC lower strand: ! TmKeller for 100 mM NaCl, zero formamide

m W rp K ! Edapters for cleavage n (ON HCFR36-1) 5'-agttctcccTGCAgctgaactc-3' 68.0 64.5 (0N_HCFR36-1A) 5'-ttctcccTGCAgctgaactc-3' 62.0 62.5 (0N_HCFR36-1B) 5'-ttctcccTGCAgctgaac-3' 56.0 59.9 (0N_HCFR33-15) 5'-cgctgtatcTGCAaatgaacag-3' 64.0 60.8 (0N_HC FR3 3-15A) 5'-ctgtatcTGCAaatgaacag-3' 56.0 56.3 (0N_HCFR33-15B) 5'-ctgtatcTGCAaatgaac-3' 50.0 53.1 (ON HCFR33-11) 5'-cactgtatcTGCAaatgaacag-3’ 62.0 58.9 (ON_HCFR35-51) 5’-ccgcctaccTGCAgtggagcag-3· 74.0 70.1 1 B.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned i Xbal... !D323* cgCttcacTaag tcT aaa gac aaC tcT aag aaT acT etc taC ! scab........ designed gene 3-23 gene................

I

! HpyCH4V ! .... Aflll... ! Ttg caG atg aac aac TtA aaG . . . i B.3 Extender and Bridges ! Extender (bottom strand):

I (ON_HCHpyEx01) 5 ' -cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3 ' ! ON HCHpyExOl is the reverse complement of ! 5'-cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC Ttg -3'

J ! Bridges (top strand, 9-base overlap):

I (ON_HCHpyBr016-l) 5’-cgCttcacTaag tcT aga gac aaC tcT aag- aaT acT ctC taC Ttg CAgctgaac-3' {3'-term C is blocked) i ! 3-15 et al. + 3-11 (ON_HCHpyBr023-15) 5'-cgCttcacTaag tcT aga gac aaC tcT aag- aaT acT ctC taC Ttg CAaatgaac-3' {3'-term C is blocked) 1 ! 5-51 (ON_HCHpyBr045-51) 5'-cgCttcacTaag tcT aga gac aaC tcT aag- aaT acT ctC taC Ttg CAgtggagc-3' (3'-term C is blocked}

I ! PCR primer (top strand) | (ON_HCHpyPCR) 5'-cgCttcacTaag tcT aga gac-3' t C: BlpI Probes from human HC GLGs 1 1-58,1-03,1-08,1-69,1-24,1-45,1-4 6,1-f, 1-e acatggaGCTGAGCagcctgag 2 1-02 acatggaGCTGAGCaggctgag 3 1-18 acatggagctgaggagcctgag 4 5-51,5-a acctgcagtggagcagcctgaa 5 3-15,3-73,3-49,3-72 atctgcaaatgaacagcctgaa 6 3303,3-33,3-07,3-11,3-30, 3-21,3-23,3305, 3-4 8 atctgcaaatgaacagcctgag 7 3-20,3-74,3-09,3-43 atctgcaaatgaacagtctgag 8 14-1 atctgcagatctgcagcctaaa 9 · 3-66,3-13,3-53,3-d atcttcaaatgaacagcctgag 10 3-64 atcttcaaatgggcagcctgag 11 4301,4-28,4302,4-04,4304,4-31,4-34,4-39,4-59, 4-61,4-b ccctgaaGCTGAGCtctgtgac 12 6-1 ccctgcagctgaactctgtgac 13 2-70,2-05 tccttacaatgaccaacatgga 14 2-26 tccttaccatgaccaacatgga D: BlpI REdaptors, Extenders, and Bridges D.1 REdaptors

T„H TmK (BlpF3HCl-58) 5'-ac atg gaG CTG AGC age ctg ag-3' 70 66. 4 (BlpF3HC6-l) 5'-cc ctg aag ctg age tet gtg ac-3’ 70 66. 4 ! BlpF3HC6-l matches 4-30.1, not 6-1. D.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned

I

BlpI ! Xbal... !D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT etc taC Ttg caG atg aac

I ! Aflll...

! aaC TTR AGG D.3 Extender and Bridges ! Bridges (BlpF3Brl) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG-taC Ttg caG Ctg a IGC age ctg-3' (BlpF3Br2) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG-taC Ttg caG Ctg a|gc tet gtg-3' ! | lower strand is cut here ! Extender (BlpF3Ext) 5'-TcAgcTgcAAaTAcAAAgTATTTTTAcTqTTATcTcTAgA cTqAqTqAAqcq- 3 1 ! BlpF3Ext is the reverse complement of: ! 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3' i (BlpF3PCR) 5'-cgCttcacTcag tcT aga gaT aaC-3' E: HpyCH4III Distinct GLG sequences surrounding site, bases 77-98 1 102#l,118#4,146#7,169#9,le#10,311#17,353#30,404#37,4301 ccgtgtattactgtgcgagaga 2 103#2,307((15, 321021, 3303#24,333#2 6, 348#28,364 #31, 366032 ctgtgtattactgtgcgagaga 3 108#3 ccgtgtattactgtgcgagagg 4 12405, If»11 ccgtgtattactgtgcaacaga 5 14506 ccatgtattactgtgcaagata 6 15808 ccgtgtattactgtgcggcaga 7 205012 ccacatattactgtgcacacag 8 226013 ccacatattactgtgcacggat 9 270*14 ccacgtattactgtgcacggat 10 309#16,343#27 ccttgtattactgtgcaaaaga u 313*18,374*35,61*50 ctgtgtattactgtgcaagaga 12 315#19 ccgtgtattactgtaccacaga 13 320#20 ccttgtatcactgtgcgagaga 14 323*22 ccgtatattactgtgcgaaaga 15 330*23,3305*25 ctgtgtattactgtgcgaaaga 16 349*29 ccgtgtattactgtactagaga 17 372*33 ccgtgtattactgtgctagaga 18 3 73413 4 ccgtgtattactgtactagaca 19 3d#36 ctgtgtattactgtaagaaaga 20 428#38 ccgtgtattactgtgcgagaaa 21 4302*40,4304#41 ccgtgtattactgtgccagaga 22 439444 ctgtgtattactgtgcgagaca 23 551*48 ccatgtattactgtgcgagaca 24 5a#49 ccatgtattactgtgcgaga __ F: HpyCH4III KEdaptors, Extenders, and Bridges F.1 REdaptors ! ONs for cleavage of HC(lower) in FR3(bases 77-97) ! For cleavage with HpyCH4III, Bst4CI, or Taal ! cleavage is in lower chain before base 88. I 77 788 888 888 889 999 999 9

! 78 901 234 567 890 123 456 7 TmM fp K J-m (H43.77.97.1 — 02#1) 5'-cc gtg tat tAC TGT gcg aga g-3’ 6462.6 (H43.77.97,l-03#2) 5'-c§j gtg tat tAC TGT gcg aga g-3' 6260.6 (H43.77.97.108#3) 5'-cc gtg tat tAC TGT gcg aga g-3' 6462.6 {H43.77.97.323#22) 5'-cc gt| tat tac tgt gcg ala g-3' 6058.7 (H43.77.97.330123) 5’-cf gtg tat tac tgt gcg ala g-3' 6058.7 (H4 3.77.97.439#44 ) 5 ' -cjS gtg tat tac tgt gcg aga §-3' 6260.6 (H4 3.77.97.551#48 ) 5 '-cc atg tat tac tgt gcg aga 1-3' 6260.6 (H43.77.97.5a#49) 5 '-cc Stg tat tAC TGT gcg aga f-3' 5858.3 F. 2 Extender and Bridges ! Xbal and Aflll sites in bridges are bunged (H43.XABrl) 5'-ggtgtagtga- | TCT | AGt | gac | aac | tct | aag I aat | act | etc | tac | ttg I cag I atg I -I aacl aqC I TTtIAGqI get Igaqlgac I aCT I GCA I Gtc I tac I tat tgt gcg aga-3' (H4 3.XABr2) 5'-ggtgtagtga- | TCT | AGt | gac | aac | tct | aag | aat | act | etc | tac | ttg | cag | atg | - I aac I aqC I TTt I AGq I get I gag I gac I aCT I GCA I Gtc I tac 1 tat tgt gcg aaa-3' (H43.XAExt) 5'-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc TgcAAgTAgA- gAgTATTeTT AgAgTTgTcT cTAgATcAcT AcAcc-3' !H43.XAExt is the reverse complement of ! 5'-ggtgtagtga- ! | TCT | AGA | gac | aac | tct | aag | aat | act | ctc | tac | ttg | cag | atg | - « I aac I acC I TTAI AGq I act I gaql aac I aCT I GCA I Gtc 1 tac I tat -3' (H43.XAPCR) 5'-ggtgtagtga |TCT|AGA|gac|aac-3' ! Xbal and Aflll sites in bridges are bunged (H43.ABrl) 5'-ggtgtagtga- I aac I acC I TTt I AGq I get I gac I gac I aCT I GCA I Gtc I tac It at tgt gcg aga-3' (H43.ABr2) 5'-ggtgtagtga- I aac I agC I TTt I AGq I get I gag I gac I aCT I GCA I Gtc I tac I tat tgt gcg aaa-3' (H4 3 . AExt) 5 ' -ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3 ' ! (H43.AExt) is the reverse complement of 5' -ggtgtagtga-* I aac I agC I TTAI AGq I get I gag I gac I aCT I GCA I Gtc I tac I tat -3' (H4 3.APCR) 5 '-ggtgtagtga I aac I aqC I TTA I AGq I get I q-3 1

Table 5: Analysis of frequenq' of matching REdaptors in actual V genes A: HpyCH4V in HC at bases 35-56

Number of mismatches..................... Number

Tri Ntot 0 1 2 3 4 5 6 7 8_9 IQ.., Cut-Id-Probe- 1 510 5 11 274 92 61 25 22 11 1 3 5 443 6-1 agttctcccTGCAgctgaactc 2 192 54 42 32 24 15 2 3 10 3 1 6 167 3-11 cactgtatcTGCAaatgaacag 3 58 19 7 17 6 5 1 0 1 0 2 0 54 3-09 ccctgtatcTGCAaatgaacag 4 267 42 33 9 Θ 8 82 43 22 8 11 1 100 5-51 ccgcctaccTGCAgtggagcag 5 250 111 59 41 24 7 5 1 0 0 2 0 242 3-15 cgctgtatcTGCAaatgaacag 6 7 02010000040 3 7-4.1 cggcatatcTGCAgatctgcag 7 η 02200210000 4 3-73 cggcgtatcTGCAaatgaacag 8 26 10 4 1 3 1 2 1 3 1 0 0 19 5-a ctgcctaccTGCAgtggagcag g 21 82316100000 20 3-49 tcgcctatcTGCAaatgaacag 1338 249 162 379 149 103 120 71 47 13 23 12 1052 249 411 790 939 1162 1280 1316 ____1042 1233 1293 1338_

Id_Probe_ dotted probe--- 6-1 agttctcccTGCAgctgaactc agttctcccTGCAgctgaactc 3-11 cactgtatcTGCAaatgaacag cac.g.at.....aa.....ag 3-09 ccctgtatcTGCAaatgaacag ccc.g.at.....aa.....ag 5-51 ccgcctaccTGCAgtggagcag ccgc..a.......tg..g.ag 3-15 cgctgtatcTGCAaatgaacag c.c.g.at.....aa.....ag 7-4.1 cggcatatcTGCAgatctgcag c.gca.at......a.ctg.ag 3-73 cggcgtatcTGCAaatgaacag c.gcg.at.....aa.....ag 5-a ctgcctaccTGCAgtggagcag ctgc..a.......tg..g.ag 3-49 tcgcctatcTGCAaatgaacag tcgc..at.....aa.....ag

Seqs with the expected RE site only.......1004 (Counts only cases with 4 or fewer mismatches)

Seqs with only an unexpected site......... 0

Seqs with both expected and unexpected.... 48 (Counts only cases with 4 or fewer mismatches)

Seqs with no sites........................ 0

B: Blpl in 11C

Id Ntot 012345678 Ncut Name_ 1 133 73 16 11 13 6 9 1 4 0 119 1-5Θ acatggaGCTGAGCagcctgag 2 14 11 1 0 0 0 0 1 0 1 .12 1-02 acatggagctgagcaggctgag 3 34 17 82610000 0 1-18 acatggagctgaggagcctgag 4 120 50 32 16 10 9 1 1 1 0 2 5-51 acctgcagtggagcagcctgaa 5 55 13 11 10 17 3 1 0 0 0 0 3-15 atctgcaaatgaacagcctgaa 6 340 186 88 41 15 6 3 0 1 0 0 3303 atctgcaaatgaacagcctgag 7 82 25 16 25 12 1 3 0 0 0 0 3-20 atctgcaaatgaacagtctgag 0 3 020100000 074.1 atctgcagatctgcagcctaaa 9 23 18 22100000 0 3-66 atcttcaaatgaacagcctgag 10 2 101000000 0 3-64 atcttcaaatgggcagcctgag 11 486 249 78 81 38 21 10 4 4 1 467 4301 ccetgaagctgagctctgtgac 12 i6 631011310 16-1 ccctgcagctgaactctgtgac 13 28 15 82210000 0 2-70 tccttacaatgaccaacatgga 14 2 020000000 0 2-26 tccttaccatgaccaacatgga 601

Name Full sequence Dot mode 1-58 acatggaGCTGAGCagcctgag acatggaGCTGAGCagcctgag 1-02 acatggagctgagcaggctgag ................g..... 1- 18 acatggagctgaggagcctgag .............g........ 5- 51 acctgcagtggagcagcctgaa . .c..c..tg...........a 3-15 atctgcaaatgaacagcctgaa . tc. .c. aa . . . a........a 3-30.3 atctgcaaatgaacagcctgag .tc..c.aa... a......... 3-20 atctgcaaatgaacagtctgag .tc..c.aa... a...t..... 7-4.1 atctgcagatctgcagcctaaa .tc..c..a.ct.......a.a 3-66 atcttcaaatgaacagcctgag .tc.tc.aa... a......... 3- 64 atcttcaaatgggcagcctgag ,tc.tc.aa..g.......... 4- 30.1 ccctgaagctgagctctgtgac c.c..a........tctg...c 6- 1 ccctgcagctgaactctgtgac c.c..c......a.tctg...c 2- 70 tccttacaatgaccaacatgga t.c.tacaa...c.. a. a..ga 2-26 tccttaccatgaccaacatgga t.c.tacca...c. . a . a..ga

Seqs with the expected RE site only....... 597 (counting sequences with 4 or fewer mismatches)

Seqs with only an unexpected site......... 2

Seqs with both expected and unexpected.... 2

Seqs with no sites........................ 686

C: HpyCHUIil, Bst4CI, or Taal in I IC

In scoring whether the RE site of interest is present, only ONs that have 4 or fewer mismatches are counted. Number of sequences.......... 1617

Group 337 471 363 218 130 58 23 11 6

Cumulative 337 808 1171 1389 1519 1577 1600 1611 1617

Seqs with the expected RE site only.......1511

Seqs with only an unexpected site......... 0

Seqs with both expected and unexpected.... 8 Seqs with no sites........................ 0

Table 5D:

Analysis repeated using only 8 best REdaptors Id Ntot 01234567 8 + 1 301 78 101 54 32 16 9 10 1 0 281 102#1 ccgtgtattactgtgcgagaga 2 493 69 155 125 73 37 14 11 3 6 459 103#2 ctgtgtattactgtgcgagaga 3 189 52 45 38 23 18 5 4 13 176 108#3 ccgtgtattactgtgcgagagg 4 127 29 23 28 24 10 6 5 2 0 114 323#22 ccgtatattactgtgcgaaaga 5 78 21 25 14 11 1 4 2 0 0 72 330#23 ctgtgtattactgtgcgaaaga 6 79 15 17 25 8 11 1 2 0 0 76 439#44 ctgtgtattactgtgcgagaca 7 43 14 15 5 5 3 0 1 0 0 42 551#48 ccatgtattactgtgcgagaca 8 307 26 63 72 51 38 24 14 13 6 250 5a#49 ccatgtattactgtgcgaga 1 102#1 ccgtgtattactgtgcgagaga ccgtgtattactgtgcgagaga 2 103#2 ctgtgtattactgtgcgagaga .t.................... 3 108#3 ccgtgtattactgtgcgagagg .....................g 4 323#22 ccgtatattactgtgcgaaaga ....a.............a... 5 330#23 ctgtgtattactgtgcgaaaga -t................a... 6 439#44 ctgtgtattactgtgcgagaca .t..................c. 7 551#48 ccatgtattactgtgcgagaca ..a.................c.

8 5a#49 ccatgtattactgtgcgagaAA . .a.................AA

Seqs with the expected RE site only.......1463 / 1617

Seqs with only an unexpected site......... 0

Seqs with both expected and unexpected.... 7 Seqs with no sites........................ 0

Table 6: Human HC GLG FR1 Sequences VH Exon - Nucleotide sequence alignment VH1

1-02 CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC

TCC TGC AAG GCT TCT GGA TAC ACC TTC ACC

1-03 cag gtC cag ctT gtg cag tct ggg get gag gtg aag aag cct ggg gcc tea gtg aag gtT

tcc tgc aag get tct gga tac acc ttc acT 1-08 cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg gcc tea gtg aag gtc tcc tgc aag get tct gga tac acc ttc acc 1-18 cag gtT cag ctg gtg cag tct ggA get gag gtg aag aag cct ggg gcc tea gtg aag gtc tcc tgc aag get tct ggT tac acc ttT acc 1-24 cag gtc cag ctg gtA cag tct ggg get gag gtg aag aag cct ggg gcc tea gtg aag gtc

tcc tgc aag gTt teC gga.tac acc etc acT

1-45 cag Atg cag ctg gtg cag tct ggg get gag gtg aag aag Act ggg Tcc tea gtg aag gtT tcc tgc aag get teC gga tac acc ttc acc

1-46 cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg gcc tea gtg aag gtT tcc tgc aag gcA tct gga tac acc ttc acc 1-58 caA Atg cag ctg gtg cag tct ggg Cct gag gtg aag aag cct ggg Acc tea gtg aag gtc

tcc tgc aag get tct gga tTc acc ttT acT 1-69 cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg Tcc teG gtg aag gtc tcc tgc aag get tct gga GGc acc ttc aGe 1-e cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg Tcc teG gtg aag gtc tcc tgc aag get tct gga GGc acc ttc aGe , 1- f Gag gtc cag ctg gtA cag tct ggg get gag gtg aag aag cct ggg geT Aca gtg aaA Ate tcc tgc aag gTt tct gga tac acc ttc acc VH2

2- 05 CAG ATC ACC TTG AAG GAG TCT GGT CCT ACG CTG GTG AAA CCC ACA CAG ACC CTC ACG CTG

ACC TGC ACC TTC TCT GGG TTC TCA CTC AGC 2-26 cag Gtc acc ttg aag gag tct ggt cct GTg ctg gtg aaa ccc aca Gag acc etc aeg ctg acc tgc acc Gtc tct ggg ttc tea etc age 2- 70 cag Gtc acc ttg aag gag tct ggt cct Geg ctg gtg aaa ccc aca cag acc etc acA ctg acc tgc acc ttc tct ggg ttc tea etc age VH3

3- 07 GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTG GTC CAG CCT GGG GGG TCC CTG AGA CTC

TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT 3-09 gaA gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggC Agg tcc ctg aga etc tcc tgt gca gcc tct gga ttc acc ttt GAt 3-11 Cag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc Aag cct ggA ggg tcc ctg aga etc tcc tgt gca gcc tct gga ttc acc ttc agt 3-13 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga etc tcc tgt gca gcc tct gga ttc acc ttc agt 3-15 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA Aag cct ggg ggg tcc ctT aga etc tcc tgt gca gcc tct gga ttc acT ttc agt 3-20 gag gtg cag ctg gtg gag tct ggg gga ggT Gtg gtA cGg cct ggg ggg tcc ctg aga etc tee tgt gca gee tct gga ttc ace ttt GAt 3-21 gag gtg cag ctg gtg gag tct ggg gga ggc Ctg gtc Aag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttC agt 3-23 gag gtg cag ctg Ttg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tec ctg aga etc

tec tgt gca gee tct gga ttc acc ttt agC 3-30 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttc agt 3-30.3 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttC agt 3-30.5 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttC agt 3-33 Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tec ctg aga etc tec tgt gca geG tct gga ttc acc ttc agt 3-43 gaA gtg cag ctg gtg gag tct ggg gga gTc Gtg gtA cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttt GAt 3-48 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttc agt 3-49 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag ccA ggg Cgg tec ctg aga etc tec tgt Aca geT tct gga ttc acc ttt Ggt 3-53 gag gtg cag ctg gtg gag Act ggA gga ggc ttg Ate cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct ggC ttc acc GtC agt 3-64 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttC agt 3-66 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc GtC agt 3-72 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggA ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttc agt 3-73 gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tec ctg aAa etc tec tgt gca gee tct ggG ttc acc ttc agt 3-74 gag gtg cag ctg gtg gag teC ggg gga ggc ttA gtT cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc ttc agt 3- d gag gtg cag ctg gtg gag tct Cgg gga gTc ttg gtA cag cct ggg ggg tec ctg aga etc tec tgt gca gee tct gga ttc acc GtC agt VH4

4- 04 CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG GGG ACC CTG TCC CTC

ACC TGC GCT GTC TCT GGT GGC TCC ATC AGC 4-28 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct teg gAC acc ctg tcc etc acc tgc get gtc tct ggt TAc tcc ate age 4-30.1 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc etc acc tgc Act gtc tct ggt ggc tcc ate age 4-30.2 cag Ctg cag ctg cag gag teC ggc Tea gga ctg gtg aag cct tcA CAg acc ctg tcc etc acc tgc get gtc tct ggt ggc tcc ate age 4-30.4 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tee ct ace tgc Act gtc tet ggt ggc tee ate age 4-31 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tee ct< acc tgc Act gtc tet ggt ggc tee ate age 4-34 cag gtg cag ctA cag Cag tGg ggc Gca gga ctg Ttg aag cct teg gAg acc ctg tee ct<

acc tgc get gtc tAt ggt ggG tee Ttc agT 4-39 cag Ctg cag ctg cag gag teg ggc cca gga ctg gtg aag cct teg gAg acc ctg tee ct< acc tgc Act gtc tet ggt ggc tee ate age 4-59 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct teg gAg acc ctg tee eti

acc tgc Act gtc tet ggt ggc tee ate agT 4-61 cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct teg gAg acc ctg tee eti acc tgc Act gtc tet ggt ggc tee Gtc age 4- b cag gtg cag ctg cag gag teg ggc cca gga ctg gtg aag cct teg gAg acc ctg tee eti acc tgc get gtc tet ggt TAc tee ate age VH5

5- 51 GAG GTG CAG CTG GTG CAG TCT GGA GCA GAG GTG AAA AAG CCC GGG GAG TCT CTG AAG ATI

TCC TGT AAG GGT TCT GGA TAC AGC TTT ACC 5- a gaA gtg cag ctg gtg cag tet gga gca gag gtg aaa aag ccc ggg gag tet ctg aGg at< tee tgt aag ggt tet gga tac age ttt acc VH6 6- 1 CAG GTA CAG CTG CAG CAG TCA GGT CCA GGA CTG GTG AAG CCC TCG CAG ACC CTC TCA CT'

ACC TGT GCC ATC TCC GGG GAC AGT GTC TCT VH7 7- 4.1 CAG GTG CAG CTG GTG CAA TCT GGG TCT GAG TTG AAG AAG CCT GGG GCC TCA GTG AAG GT'

TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT

Table 7: RERS sites in Human HC GLG FRls where there are at least 20 GLGs cut Bsgl GTGCAG 71 (cuts 16/14 bases to tight) 1: 4 1: 13 2: 13 3: 4 3: 13 4: 13 6: 13 7: 4 7: 13 8: 13 9: 4 9: 13 10: 4 10: 13 15: 4 15: 65 16: 4 16: 65 17: 4 17: 65 18: 4 18: 65 19: 4 19: 65 20: 4 20: 65 21: 4 21: 65 22: 4 22: 65 23: 4 23: 65 24: 4 24: 65 25: 4 25: 65 26: 4 26: 65 27: 4 27: 65 28: 4 28: 65 29: 4 30: 4 30: 65 31: 4 31: 65 32: 4 32: 65 33: 4 33: 65 34: 4 34: 65 35: 4 35: 65 36: 4 36: 65 37: 4 38: 4 39: 4 41: 4 42: 4 43: 4 45: 4 46: 4 47: 4 48: 4 48: 13 49: 4 49: 13 51: 4

There are 39 hits at base# 4 There are 21 hits at base# 65 ctgcac 9 12: 63 13: 63 14: 63 39: 63 41: 63 42: 63 44: 63 45: 63 46: 63

Bbvl GCAGC 65 1: 6 3: 6 6: 6 7: 6 8: 6 9: 6 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 36: 6 36: 67 37: 6 38: 6 39: 6 40: 6 41: 6 42: 6 43: 6 44: 6 45: 6 46: 6 47: 6 48: 6 49: 6 50: 12 51: 6

There are 43 hits at base# 6 Bolded sites very near sites listed below

There are 21 hits at base# 67 gctgc 13 37: 9 38: 9 39: 9 40: 3 40: 9 41: 9 42: 9 44: 3 44: 9 45: 9 46: 9 47: 9 50: 9

There are 11 hits at base# 9

BsoFI GCngc 78 1: 6 3: 6 6: 6 7: 6 8: 6 9: 6 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 36: 6 36: 67 37: 6 37: 9 38: 6 38: 9 39: 6 39: 9 40: 3 40: 6 40: 9 41: 6 41: 9 42: 6 42: 9 43: 6 44: 3 44: 6 44: 9 45: 6 45: 9 46: 6 46: 9 47: 6 47: 9 48: 6 49: 6 50: 9 50: 12 51: 6

There are 43 hits at base# 6 These often occur together.

There are 11 hits at base# 9

There are 2 hits at base# 3

There are 21 hits at base# 67

Tsel Gcwgc 78 1:6 3:6 6:6 7:6 8:6 9:6 i 10: 6 15: 6 15: 67 16: 6 16: 67 17: 6 17: 67 18: 6 18: 67 19: 6 19: 67 20: 6 20: 67 21: 6 21: 67 22: 6 22: 67 23: 6 23: 67 24: 6 24: 67 25: 6 25: 67 26: 6 26: 67 27: 6 27: 67 28: 6 28: 67 29: 6 I 30: 6 30: 67 31: 6 31: 67 32: 6 32: 67 33: 6 33: 67 34: 6 34: 67 35: 6 35: 67 36: 6 36: 67 37: 6 37: 9 38: 6 38: 9 39: 6 39: 9 40: 3 40: 6 40: 9 41: 6 41: 9 42: 6 42: 9 43: 6 44: 3 44: 6 j 44: 9 45: 6 4 5: 9 46: 6 46: 9 47: 6 47: 9 48: 6 49: 6 50: 9 50: 12 51: 6

There are 43 hits at base# 6 Often together.

There are 11 hits at base# 9

There are 2 hits at base# 3 There are 1 hits at base# 12

There are 21 hits at base# 67

MspAlI CMGckg 48 1: 7 3: 7 4: 7 5: 7 6: 7 7: 7 8: 7 9: 7 10: 7 11: 7 15: 7 16: 7 17: 7 18: 7 19: 7 20: 7 21: 7 22: 7 23: 7 24: 7 25: 7 26: 7 27: 7 28: 7 29: 7 30: 7 31: 7 32: 7 33: 7 34: 7 35: 7 36: 7 37: 7 38: 7 39: 7 40: 1 40: 7 41: 7 42: 7 44: 1 44: 7 45: 7 46: 7 47: 7 48: 7 49: 7 50: 7 51: 7

There are 46 hits at base# 7

PvuII CAGctg 48 1: 7 3: 7 4: 7 5: 7 6: 7 7: 7 8: 7 9: 7 10: 7 11: 7 15: 7 16: 7 17: 7 18: 7 19: 7 20: 7 21: 7 22: 7 23: 7 24: 7 25: 7 26: 7 27: 7 28: 7 29: 7 30: 7 31: 7 32: 7 33: 7 34: 7 35: 7 36: 7 37: 7 38: 7 39: 7 40: 1 40: 7 41: 7 42: 7 44: 1 44: 7 45: 7 46: 7 47: 7 48: 7 49: 7 50: 7 51: 7

There are 46 hits at base# 7 There are 2 hits at base# 1

Alul AGct 54 1: 8 2: 8 3: 8 4: 8 4: 24 5: 8 6: 8 7: 8 8: 8 9: 8 10: 8 11: 8 15: 8 16: 8 17: 8 18: 8 19: 8 20: 8 21: 8 22: 8 23: 8 24: 8 25: 8 26: 8 27: 8 28: 8 29: 8 29: 69 30: 8 31: 8 32: 8 33: 8 34: 8 35: 8 36: 8 37: 8 38: 8 39: 8 40: 2 40: 8 41: 8 42: 8 43: 8 44: 2 44: 8 45: 8 46: 8 47: 8 48: 8 48: 82 49: 8 49: 82 50: 8 51: 8

There are 48 hits at base# Θ

There are 2 hits at base# 2

Ddel Ctnag 48 1:-26 1: 48 2: 26 2: 48 3: 26 3: 48 4: 26 4: 48 5: 26 5: 48 6: 26 6: 48 7: 26 7: 48 8: 26 8: 48 9: 26 10: 26 11: 26 12: 85 13: 85 14: 85 15: 52 16: 52 17: 52 18: 52 19: 52 20: 52 21: 52 22: 52 23: 52 24: 52 25: 52 26: 52 27: 52 28: 52 29: 52 30: 52 31: 52 32: 52 33: 52 35: 30 35: 52 36: 52 40: 24 49: 52 51: 26 51: 48

There are 22 hits at base# 52 52 and 48 never together.

There are 9 hits at base# 48

There are 12 hits at base# 26 26 and 24 never together.

HphI tcacc 42 1: 86 3: 86 6: 86 7: 86 8: 80 11: 86 12: 5 13: 5 14: 5 15: 80 16: 80 17: 80 18: 80 20: 80 21: 80 22: 80 23: 80 24: 80 25: 80 26: 80 27: 80 28: 80 29: 80 30: 80 31: 80 32: 80 33: 80 34: 80 35: 80 36: 80 37: 59 38: 59 39: 59 40: 59 41: 59 42: 59 43: 59 44: 59 45: 59 46: 59 47: 59 50: 59

There are 22 hits at base# 80 80 and 86 never together

There are 5 hits at base# 86

There are 12 hits at base# 59

BssKI Nccngg 50 1: 39 2: 39 3: 39 4: 39 5: 39 7: 39 8: 39 9: 39 10: 39 11: 39 15: 39 16: 39 17: 39 18: 39 19: 39 20: 39 21: 29 21: 39 . 22: 39 23: 39 24: 39 25: 39 26: 39 27: 39 28: 39 29: 39 30: 39 31: 39 32: 39 33: 39 34: 39 35: 19 35: 39 36: 39 37: 24 38: 24 39: 24 41: 24 42: 24 44: 24 45: 24 46: 24 47: 24 48: 39 48: 40 49: 39 49: 40 50: 24 50: 73 51: 39

There are 35 hits at base# 39 39 and 40 together twice.

There are 2 hits at base# 40

BsaJI Ccnngg 47 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 8: 40 9: 40 9: 47 10: 40 10: 47 11: 40 15: 40 18: 40 19: 40 20: 40 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 28: 40 29: 40 30: 40 31: 40 32: 40 34: 40 35: 20 35: 40 36: 40 37: 24 38: 24 39: 24 41: 24 42: 24 44: 24 45: 24 46: 24 47: 24 48: 40 48: 41 49: 40 49: 41 50: 74 51: 40

There are 32 hits at base# 40 40 and 41 together twice

There are 2 hits at base# 41

There are 9 hits at base# 24

There are 2 hits at base# 47

BstNI CCwgg 44

PspGI ccwgg

ScrFI(SM.Hpall) CCwgg 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 8: 40 9: 40 10: 40 11: 40 15: 40 16: 40 17: 40 18: 40 19: 40 20: 40 21: 30 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 28: 40 29: 40 30: 40 31: 40 32: 40 33: 40 34: 40 35: 40 36: 40 37: 25 38: 25 39: 25 41: 25 42: 25 44: 25 45: 25 46: 25 47: 25 50: 25 51: 40

There are 33 hits at base# 40

ScrFI CCngg 50 1: 40 2: 40 3: 40 4: 40 5: 40 7: 40 8: 40 9: 40 10: 40 11: 40 15: 40 16: 40 17: 40 18: 40 19: 40 20: 40 21: 30 21: 40 22: 40 23: 40 24: 40 25: 40 26: 40 27: 40 28: 40 29: 40 30: 40 31: 40 32: 40 33: 40 34: 40 35: 20 35: 40 36: 40 37: 25 38: 25 39: 25 41: 25 42: 25 44: 25 45: 25 46: 25 47: 25 48: 40 48: 41 49: 40 49: 41 50: 25 50: 74 51: 40

There are 35 hits at base# 40

There are 2 hits at base# 41

EcoO109I RGgnccy 34 1: 43 2: 43 3: 43 4: 43 5: 43 6: 43 7: 43 8: 43 9: 43 10: 43 15: 46 16: 46 17: 46 18: 46 19: 46 20: 46 21: 46 22: 46 23: 46 24: 46 25: 46 26: 46 27: 46 28: 46 30: 46 31: 46 32: 46 33: 46 34: 46 35: 46 36: 46 37: 46 43: 79 51: 43

There are 22 hits at base# 46 46 and 43 never together

There are 11 hits at base# 43

NlalV GGNncc 71 1: 43 2: 43 3: 43 4: 43 5: 43 6: 43 7: 43 8: 43 9: 43 9: 79 10: 43 10: 79 15: 46 15: 47 16: 47 17: 46 17: 47 18: 46 . 18: 47 19: 46 19: 47 20: 46 20: 47 21: 46 21: 47 22: 46 22: 47 23: 47 24: 47 25: 47 26: 47 27: 46 27: 47 28: 46 28: 47 29: 47 30: 46 30: 47 31: 46 31: 47 32: 46 32: 47 33: 46 33: 47 34: 46 34: 47 35: 46 35: 47 36: 46 36: 47 37: 21 37: 46 37: 47 37: 79 38: 21 39: 21 39: 79 40: 79 41: 21 41: 79 42: 21 42: 79 43: 79 44: 21 44: 79 45: 21 45: 79 46: 21 46: 79 47: 21 51: 43

There are 23 hits at base# 47 46 &amp; 47 often together There are 17 hits at base# 46 There are 11 hits at base# 43 Sau96I Ggncc 70 1: 44 2: 3 2: 44 3: 44 4: 44 5: 3 5: 44 6: 44 7: 44 8: 22 8: 44 9: 44 10: 44 11: 3 12: 22 13: 22 14: 22 15: 33 15: 47 16: 47 17: 47 18: 47 19: 47 20: 47 21: 47 22: 47 23: 33 23: 47 24: 33 24: 47 25: 33 25: 47 26: 33 26: 47 27: 47 28: 47 29: 47 30: 47 31: 33 31: 47 32: 33 32: 47 33: 33 33: 47 34: 33 34: 47 35: 47 36: 47 37: 21 37: 22 37: 47 38: 21 38: 22 39: 21 39: 22 41: 21 41: 22 42: 21 42: 22 43: 80 44: 21 44: 22 45: 21 45: 22 46: 21 46: 22 47: 21 47: 22 50: 22 51: 44

There are 23 hits at base# 47 These do not occur together. There are 11 hits at base# 44

There are 14 hits at base# 22 These do occur together.

There are 9 hits at base# 21

BsraAI GTCTCNnnnn 22 1: 58 3: 58 4: 58 5: 58 8: 58 9: 58 10: 58 13: 70 36: 18 37: 70 38: 70 39: 70 40: 70 41: 70 42: 70 44: 70 45: 70 46: 70 47: 70 48: 48 49: 48 50: 85

There are 11 hits at base# 70

Nnnnnngagac 27 13: 40 15: 48 16: 48 17: 48 18: 48 20: 48 21: 48 22: 48 23: 48 24: 48 25: 48 26: 48 27: 48 28: 48 29: 48 30: 10 30: 48 31: 48 32: 48 33: 48 35: 48 36: 48 43: 40 44: 40 45: 40 46: 40 47: 40

There are 20 hits at base# 48

Avail Ggwcc 44

Sau96I($M.HaeIII) Ggwcc 44 2: 3 5: 3 6: 44 8: 44 9: 44 10: 44 11: 3 12: 22 13: 22 14: 22 15: 33 15: 47 16: 47 17: 47 18: 47 19: 47 20: 47 21: 47 22: 47 23: 33 23: 47 24: 33 24: 47 25: 33 25: 47 26: 33 26: 47 27: 47 28: 47 29: 47 30: 47 31: 33 31: 47 32: 33 32: 47 33: 33 33: 47 34: 33 34: 47 35: 47 36: 47 37: 47 43: 80 50: 22

There are 23 hits at base# 47 44 &amp; 47 never together

There are 4 hits at base# 44

PpuMI RGgwccy 27 6: 43 8: 43 9: 43 10: 43 15: 46 16: 46 17: 46 18: 46 19: 46 20: 46 21: 46 22: 46 23: 46 24: 46 25: 46 26: 46 27: 46 28: 46 30: 46 31: 46 32: 46 33: 46 34: 46 35: 46 36: 46 37: 46 43: 79

There are 22 hits at base# 46 43 and 46 never occur together.

There are 4 hits at base# 43

BsmFI GGGAC 3 8: 43 37: 46 50: 77 gtccc 33 15: 48 16: 48 17: 48 1:0 1:0 20: 48 21: 48 22: 48 23: 48 24: 48 25: 48 26: 48 27: 48 28: 48 29: 48 30: 48 31: 48 32: 48 33: 48 34: 48 35: 48 36: 48 37: 54 38: 54 39: 54 40: 54 41: 54 42: 54 43: 54 44: 54 . 45: 54 46: 54 47: 54

There are 20 hits at base# 48 There are 11 hits at base# 54

Hinfl Gantc 80 8: 77 12: 16 13: 16 14: 16 15: 16 15: 56 15: 77 16: 16 16: 56 16: 77 17: 16 17: 56 17: 77 18: 16 18: 56 18: 77 19: 16 19: 56 19: 77 20: 16 20: 56 20: 77 21: 16 21: 56 21: 77 22: 16 22: 56 22: 77 23: 16 23: 56 23: 77 24: 16 24: 56 24: 77 25: 16 25: 56 25: 77 26: 16 26: 56 26: 77 27: 16 27: 26 27: 56 27: 77 28: 16 28: 56 28: 77 29: 16 29: 56 29: 77 30: 56 31: 16 31: 56 31: 77 32: 16 32: 56 32: 77 33: 16 33: 56 33: 77 34: 16 35: 16 35: 56 35: 77 36: 16 36: 26 36: 56 36: 77 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46

There are 34 hits at base# 16

Tfil Gawtc 21 8: 77 15: 77 16: 77 17: 77 18: 77 19: 77 20: 77 21: 77 22: 77 23: 77 24: 77 25: 77 26: 77 27: 77 28: 77 29: 77 31: 77 32: 77 33: 77 35: 77 36: 77

There are 21 hits at base# 77

Mlyl GAGTC 38 12: 16 13: 16 14: 16 15: 16 16: 16 17: 16 18: 16 19: 16 20: 16 21: 16 22: 16 23: 16 24: 16 25: 16 26: 16 27: 16 27: 26 28: 16 29: 16 31: 16 32: 16 33: 16 34: 16 35: 16 36: 16 36: 26 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46

There are 34 hits at base# 16 GACTC 21 15: 56 16: 56 17: 56 18: 56 19: 56 20: 56 21: 56 22: 56 23: 56 24: 56 25: 56 26: 56 27: 56 28: 56 29: 56 30: 56 31: 56 32: 56 33: 56 35: 56 36: 56

There are 21 hits at base# 56

Plel gagtc 38 12: 16 13: 16 14: 16 15: 16 16: 16 17: 16 18: 16 19: 16 20: 16 21: 16 22: 16 23: 16 24: 16 25: 16 26: 16 27: 16 27: 26 28: 16 29: 16 31: 16 32: 16 33: 16 34: 16 35: 16 36: 16 36: 26 37: 16 38: 16 39: 16 40: 16 41: 16 42: 16 44: 16 45: 16 46: 16 47: 16 48: 46 49: 46

There are 34 hits at base# 16 gactc 21 15: 56 16: 56 17: 56 18: 56 19: 56 20: 56 21: 56 22: 56 23: 56 24: 56 25: 56 26: 56 27: 56 28: 56 29: 56 30: 56 31: 56 32: 56 33: 56 35: 56 36: 56

There are 21 hits at base# 56 AlwNI CAGNNNctg 26 15: 68 16: 68 17: 68 18: 68 19: 68 20: 68 21: 68 22: 68 23: 68 24: 68 25: 68 26: 68 27: 68 28: 68 29: 68 30: 68 31: 68 32: 68 33: 68 34: 68 35: 68 36: 68 39: 46 40: 46 41: 46 42: 46

There are 22 hits at base# 68

Table 8: Kappa FR1 GLGs 11234567 89 10 11 12

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT ! 13 14 15 16 17 18 19 20 21 22 23 GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 012

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 02

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 018

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! 08

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! A20

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! A30

AAC ATC CAG ATG ACC CAG TCT CCA TCT GCC ATG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! L14

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT

GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! LI

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! LI5

GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L4

GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! LI8

GAC ATC CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! L5

GAC ATC CAG ATG ACC CAG TCT CCA TCT TCT GTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGT ! LI 9

GAC ATC CAG TTG ACC CAG TCT CCA TCC TTC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L8

GCC ATC CGG ATG ACC CAG TCT CCA TTC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L23

GCC ATC CGG ATG ACC CAG TCT CCA TCC TCA TTC TCT GCA TCT ACA GGA GAC AGA GTC ACC ATC ACT TGT ! L9

GTC ATC TGG ATG ACC CAG TCT CCA TCC TTA CTC TCT GCA TCT ACA GGA GAC AGA GTC ACC ATC AGT TGT . L24

GCC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L11

GAC ATC CAG ATG ACC CAG TCT CCT TCC ACC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC ! L12

GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! Oil

GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! 01

GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ! A17

GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ! A1

GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC ! A18

GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC ! A2

GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! A19

GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC ! A3

GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC TCA CCT GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC ί A23

GAA ATT GTG TTG ACG CAG TCT CCA GGC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! A27

GAA ATT GTG TTG ACG CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! All

GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L2

GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! LI6

GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L6

GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L2°

GAA ATT GTA ATG ACA GAG TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA. AGA GCC ACC CTC TCC TGC ! L2

GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT GTG TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC ! B3

GAA ACG ACA CTC ACG CAG TCT CCA GCA TTC ATG TCA

GCG ACT CCA GGA GAC AAA GTC AAC ATC TCC TGC ! B

GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC ! A26

GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC *10

GAT GTT GTG ATG ACA CAG TCT CCA GCT TTC CTC TCT GTG ACT CCA GGG GAG AAA GTC ACC ATC ACC TGC ! A14

Table 9 RERS sites found in Human Kappa FRl GLGs

Table 9 RERS sites found in Human Kappa FR1 GLGs, continued

Table 9 RERS sites found in Human Kappa FR1, continued

Table 10 Lambda FR1 GLG sequences ! VL1

CAG TCT GTG CTG ACT CAG CCA CCC TCG GTG TCT GAA GCC CCC AGG CAG AGG GTC ACC ATC TCC TGT ! la

cag tct gtg ctg acG cag ccG ccc tcA gtg tct gGG gcc ccA Ggg cag agg gtc acc ate tcc tgC ! le

cag tct gtg ctg act cag cca ccc tcA gCg tct gGG

Acc ccc Ggg cag agg gtc acc ate tcT tgt ! lc

cag tct gtg ctg act cag cca ccc tcA gCg tct gGG

Acc ccc Ggg cag agg gtc acc ate teT tgt ! lg

cag tct gtg Ttg acG cag ccG ccc tcA gtg tct gCG gcc ccA GgA cag aAg gtc acc ate tcc tgC ! lb ! VL2

CAG TCT GCC CTG ACT CAG CCT CCC TCC GCG TCC GGG TCT CCT GGA CAG TCA GTC ACC ATC TCC TGC ! 2c cag tct gcc ctg act cag cct eGe tcA gTg tcc ggg tct cct gga cag tea gtc acc ate tcc tgc! 2e cag tct gcc ctg act cag cct Gcc tcc gTg teT ggg tct cct gga cag teG Ate acc ate tcc tgc ! 2a2 cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg tct cct gga cag tea gtc acc ate tcc tgc ! 2d cag tct gcc ctg act cag cct Gcc tcc gTg teT ggg tct cct gga cag teG Ate acc ate tcc tgc ! 2b2 ! VL3

TCC TAT GAG CTG ACT CAG CCA CCC TCA GTG TCC GTG TCC CCA GGA CAG ACA GCC AGC ATC ACC TGC! 3r tcc tat gag ctg act cag cca cTc tea gtg tcA gtg

Gcc cTG gga cag acG gcc agG atT acc tgT ! 3j tcc tat gag ctg acA cag cca ccc teG gtg tcA gtg tcc cca gga caA acG gcc agG ate acc tgc! 3p tcc tat gag ctg acA cag cca ccc teG gtg tcA gtg tcc cTa gga cag aTG gcc agG ate acc tgc ! 3a teT tct gag ctg act cag GAC ccT GeT gtg teT gtg

Gcc TTG gga cag aca gTc agG ate acA tgc ! 31 -_cc tat gTg ctg act cag cca ccc tea gtg tcA gtg

Gee cca gga Aag aeG gee agG atT ace tgT ! 3h tee tat gag ctg acA cag cTa ccc teG gtg tcA gtg tee cca gga cag aca gee agG ate ace tgc ! 3e tee tat gag ctg aTG cag cca ccc teG gtg tcA gtg tee cca gga cag aeG gee agG ate ace tgc ! 3m tee tat gag ctg acA cag cca Tee tea gtg tcA gtg teT ccG gga cag aca gee agG ate ace tgc ! V2-19 VL4 CTG CCT GTG CTG ACT CAG CCC CCG TCT GCA TCT GCC TTG CTG GGA GCC TCG ATC AAG CTC ACC TGC ! 4c cAg cct gtg ctg act caA TcA TeC tet geC tet geT tCC ctg gga Tee teg Gtc aag etc acc tgc ! 4a cAg eTt gtg ctg act caA TeG ccC tet geC tet gee tCC ctg gga gee teg Gtc aag etc acc tgc ! 4b ! VL5 CAG CCT GTG CTG ACT CAG CCA CCT TCC TCC TCC GCA TCT CCT GGA GAA TCC GCC AGA CTC ACC TGC ! 5e cag Get gtg ctg act cag ccG Get tee CTc teT gca tet cct gga gCa tcA gee agT etc acc tgc ! 5c cag cct gtg ctg act cag cca Tet tee CAT teT gca tet Tet gga gCa tcA gTc aga etc acc tgc ! 5b ! VL6

AAT TTT ATG CTG ACT CAG CCC CAC TCT GTG TCG GAG TCT CCG GGG AAG ACG GTA ACC ATC TCC TGC ! 6a ! VL7

CAG ACT GTG GTG ACT CAG GAG CCC TCA CTG ACT GTG TCC CCA GGA GGG ACA GTC ACT CTC ACC TGT ! 7a cag Get gtg gtg act cag gag ccc tea ctg act gtg tee cca gga ggg aca gtc act etc acc tgt ! 7b ! VL8

CAG ACT GTG GTG ACC CAG GAG CCA TCG TTC TCA GTG TCC CCT GGA GGG ACA GTC ACA CTC ACT TGT ! 8a ! VL9

CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA TCA GCC TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC ! 9a ! VL10

CAG GCA GGG CTG ACT CAG CCA CCC TCG GTG TCC AAG GGC TTG AGA CAG ACC GCC ACA CTC ACC TGC ! 10a

Table 11 RERSs found in human lambda FR1 GLGs ! There are 31 lambda GLGs Mlyl NnnnnnGACTC 25 1:6 3:6 4:6 6:6 7:6 8:6 9: 6 10: 6 11: 6 12: 6 15: 6 16: 6 20: 6 21: 6 22: 6 23: 6 23: 50 24: 6 25: 6 25: 50 26: 6 27: 6 28: 6 30: 6 31: 6

There are 23 hits at base# 6 GAGTCNNNNNn 1 26: 34

Mwol GCNNNNNnngc 20 I: 9 2: 9 3: 9 4: 9 11: 9 11: 56 12: 9 13: 9 14: 9 16: 9 17: 9 18: 9 19: 9 20: 9 23: 9 24: 9 25: 9 26: 9 30: 9 31: 9

There are 19 hits at base# 9 Hinfl Gantc 27 1: 12 3: 12 4: 12 6: 12 7: 12 8: 12 9: 12 10: 12 11: 12 12: 12 15: 12 16: 12 20: 12 21: 12 22: 12 23: 12 23: 46 23: 56 24: 12 25: 12 25: 56 26: 12 26: 34 27: 12 28: 12 30: 12 31: 12

There are 23 hits at base# 12 Plel gactc 25 1: 12 3: 12 4: 12 6: 12 7: 12 8: 12 9: 12 10: 12 11: 12 12: 12 15: 12 16: 12 20: 12 21: 12 22: 12 23: 12 23: 56 24: 12 25: 12 25: 56 26: 12 27: 12 28: 12 30: 12 31: 12

There are 23 hits at base# 12 gagtc 1 26: 34

Ddel Ctnag 32 1: 14 2: 24 3: 14 3: 24 4: 14 4: 24 5: 24 6: 14 7: 14 7: 24 8: 14 9: 14 10: 14 11: 14 11: 24 12: 14 12: 24 15: 5 15: 14 16: 14 16: 24 19: 24 20: 14 23: 14 24: 14 25: 14 26: 14 27: 14 28: 14 29: 30 30: 14 31: 14

There are 21 hits at base# 14

BsaJI Ccnngg 38 1: 23 1: 40 2: 39 2: 40 3: 39 3: 40 4: 39 4: 40 5: 39 11: 39 12: 38 12: 39 13: 23 13: 39 14: 23 14: 39 15: 38 16: 39 17: 23 17: 39 18: 23 18: 39 21: 38 21: 39 21: 47 22: 38 22: 39 22: 47 26: 40 27: 39 28: 39 29: 14 29: 39 30: 38 30: 39 30: 47 31: 23 31: 32

There are 17 hits at base# 39

There are 5 hits at base# 38

There are 5 hits at base# 40 Makes cleavage ragged.

Mnll cctc 35 1: 23 2: 23 3: 23 4: 23 5: 23 6: 19 6: 23 7: 19 8: 23 9: 19 9: 23 10: 23 11: 23 13: 23 14: 23 16: 23 17: 23 18: 23 19: 23 20: 47 21: 23 21: 29 21: 47 22: 23 22: 29 22: 35 22: 47 23: 26 23: 29 24: 27 27: 23 28: 23 30: 35 30: 47 31: 23

There are 21 hits at base# 23

There are 3 hits at base# 19

There are 3 hits at base# 29

There are 1 hits at base# 26

There are 1 hits at base# 27 These could make cleavage ragged. gagg 3 1: 48 2: 48 3: 48 4: 48 27: 44 28: 44 29: 44

BssKI Nccngg 39 1: 40 2: 39 3: 39 3: 40 4: 39 4: 40 5: 39 6: 31 6: 39 7: 31 7: 39 8: 39 9: 31 9: 39 10: 39 11: 39 12: 38 12: 52 13: 39 13: 52 14: 52 16: 39 16: 52 17: 39 17: 52 18: 39 18: 52 19: 39 19: 52 21: 38 22: 38 23: 39 24: 39 26: 39 27: 39 28: 39 29: 14 29: 39 30: 38

There are 21 hits at base# 39 There are 4 hits at base# 38 There are 3 hits at base# 31 There are 3 hits at base# 40 Ragged

BstNI CCwgg 30 1: 41 2: 40 5: 40 6: 40 7: 40 8: 40 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28: 40 29: 15 29: 40 30: 39

There are 17 hits at base# 40 There are 7 hits at base# 53 There are 4 hits at base# 39 There are 1 hits at base# 41 Ragged

PspGI ccwgg 30 1: 41 2: 40 5: 40 6: 40 7: 40 8: 40 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 53 21: 39 22: 39 23: 40 24: 40 27: 40 28: 40 29: 15 29: 40 30: 39

There are 17 hits at base# 40 There are 7 hits at base# 53

There are 4 hits at base# 39

There are 1 hits at base# 41

ScrFI CCngg 39 1: 41 2: 40 3: 40 3: 41 4: 40 4: 41 5: 40 6: 32 6: 40 7: 32 7: 40 8: 40 9: 32 9: 40 10: 40 11: 40 12: 39 12: 53 13: 40 13: 53 14: 53 16: 40 16: 53 17: 40 17: 53 18: 40 18: 53 19: 40 19: 53 21: 39 22: 39 23: 40 24: 40 26: 40 27: 40 28: 40 29: 15 29: 40 30: 39

There are 21 hits at base# 40

There are 4 hits at base# 39

There are 3 hits at base# 41

Maelll gtnac 16 1: 52 2: 52 3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 26: 52 27: 10 27: 52 28: 10 28: 52 29: 10 29: 52 30: 52

There are 13 hits at base# 52

Tsp45I gtsac 15 1: 52 2: 52 3: 52 4: 52 5: 52 6: 52 7: 52 9: 52 27: 10 27: 52 28: 10 28: 52 29: 10 29: 52 30: 52

There are 12 hits at base# 52

HphI tcacc 26 1: 53 2: 53 3: 53 4: 53 5: 53 6: 53 7: 53 8: 53 9: 53 10: 53 11: 59 13: 59 14: 59 17: 59 18: 59 19: 59 20: 59 21: 59 22: 59 23: 59 24: 59 25: 59 27: 59 28: 59 30: 59 31: 59

There are 16 hits at base# 59

There are 10 hits at base# 53

BspMI ACCTGCNNNNn 14 11: 61 13: 61 14: 61 17: 61 18: 61 19: 61 20: 61 21: 61 22: 61 23: 61 24: 61 25: 61 30: 61 31: 61

There are 14 hits at base# 61 Goes into CDR1

Table 12: Matches to URE FR3 adapters in 79 human HC. A. List of Heavy-chains genes sampled AF008566 AF103367 HSA235674 HSU94417 S83240 AF035043 AF103368 HSA235673 HSU94418 SABVH369

AF103026 AF103369 HSA240559 HSU96389 SADEIGVH

af103033 AF103370 HSCB201 HSU96391 SAH2IGVH

AF103061 af103371 HSIGGVHC HSU96392 SDA3IGVH

Af103072 AF103372 HSU44791 HSU96395 SIGVHTTD

af103078 AF158381 HSU44793 HSZ93849 SUK4IGVH AF1030 99 E05213 HSU82771 HSZ93850 AF103102 E05886 HSU82949 HSZ93851 AF103103 E05887 HSU82950 HSZ93853 AF103174 HSA235661 HSU82952 HSZ93855 AF103186 HSA235664 HSU82961 HSZ93857 af103187 HSA235660 HSU86522 HSZ93860 AF103195 HSA235659 HSU86523 HSZ93863

af103277 HSA235678 HSU92452 MCOMFRAA

af10328 6 HSA235677 HSU94412 MCOMFRVA AF103309 HSA23567 6 HSU94415 S82745 af10334 3 HSA235675 HSU94416 S82764

Table 12B. Testing all distinct GLGs from bases 89-1 to 93.2 of the heavy variable domain

Id Nb 0 1 2 3 4 SEQ ID NO: 1 38 15 11 10 0 2 Seql gtgtattactgtgc 25 2 19 76420 Seq2 gtAtattactgtgc 26 3 1 0 0 1 0 0 Seq3 gtgtattactgtAA 27 4 7 1 5 1 0 0 Seq4 gtgtattactgtAc 28 5 0 00000 Seq5 Ttgtattactgtgc 29 6 0 0 0 0 0 0 Seq6 TtgtatCactgtgc 30 7 3 10110 Seq7 ACAtattactgtgc 31 8 2 0 2 0 0 0 Seq8 ACgtattactgtgc 32 9 _9_2 2 4 1 0 Sea9 ATatattactgtgc_33

Group 262621 4 2

Cumulative 26 52 73 77 79

Table 12C Most important URE recognition seqs in FR3 Heavy 1 VHSzyl GTGtattactgtgc (ON_SHC103) (SEQ ID NO:25) 2 VHSzy2 GTAtattactgtgc (ON_SHC323) (SEQ ID N0:26) 3 VHSzy4 GTGtattactgtac (ON_SHC349) (SEQ ID NO:28) i 4 VHSzy9 ATGtattactgtgc (ON_SHC5a) (SEQ ID NO:33)

Table 12D, testing 79 human HC V genes with four probes

Number of sequences.......... 7 9

Number of bases.............. 29143

Number of mismatches Id Best 0 1 2 3 4 5 1 39 15 11 10 1 2 0 Seql gtgtattactgtgc (SEQ ID NO:25) 2 22 765301 Seq2 gtAtattactgtgc (SEQ ID NO:26) 3 7 151000 Seq4 gtgtattactgtAc (SEQ ID NO:28) 4 _ii 2 4 4 1 0 0 Sea9 ATatattactqtqc (SEQ ID NO:33)

Group 252620 5 2

Cumulative 25 51 71 76 78

One sequence has five mismatches with sequences 2, 4, and 9; it is scored as best for 2.

Id is the number of the adapter.

Best is the number of sequence for which the identified adapter was the best available.

The rest of the table shows how well the sequences match the adapters. For example, there are 10 sequences that match VHSzyl(Id=l) with 2 mismatches and are worse for all other adapters. In this sample, 90% come within 2 bases of one of the four adapters.

Table 13

The following list of enzymes was taken from http://rebase. neb.com/cai-bin/asvmmlist I have removed the enzymes that a) cut within the recognition, b) cut on both sides of the recognition, or c) have fewer than 2 bases between recognition and closest cut site. REBASE Enzymes 04/13/2001 . .

Type II restriction enzymes with asymmetric recognition sequences:

Enzymes Recognition Sequence Isoschizomers Suppliers

Aarl CACCTGCNNNNANNNN_ - y

Acelll CAGCTCNNNNNNNANNNN_ - -

Bbr7I GAAGACNNNNNNNANNNN_ - -

Bbvl GCAGCNNNNNNNNANNNN_ y

BbvII GAAGACN N A N N N N_

Bce83I CTTGAGNNNNNNNNNNNNNN_NNA - -

BceAI ACGGCNNNNNNNNNNNNA NN_ - y

BeefI ACGGCNNNNNNNNNNNNAN_ - -

BciVI G TATCCNNNNN_NΛ Bful y

Bfil ACTGGGNNNN_NA Bmrl y

BinI GGATCNNNNAN_

BscAI GCATCNNNNANN_ - "

BseRI GAGGAGNNNNNNNN_NNA - y

BsmFI GGGACNNNNNNNNNNA NNNN_ BspLUllIII y

BspMI ACCTGCNNNNANNNN_ Acc36I y .

Ecil GGCGGANNNNNNNNN_NNA - 'y

Eco57I CTGAAGNNNNNNNNNNNNNN_NNA BspKT5I y

Faul CCCGCNNNNANN_ BstFZ438I y

FokI GGATGNNNNNNNNNANNNN_ BstPZ418I y

Gsul CTGGAGNNNNNNNNNNNNNN_NNA - y

Hgal GACGCNNNNNANNNNN_ - y

HphI GGTGANNNNNNN_NA AsuHPI y

MboII GAAGANNNNNNN_NA - y

Mlyl GAGTCNNNNNA Schl y

Mmel TCC RACNNNNNNNNNNNNNNNNNN_NNΛ - -

Mnll CCTCNNNNNN_NA - y

Plel GAGTCNNNNAN_ PpsI y

RleAI CCCACANNNNNNNNN_NNNA - "

SfaNI GCATCNNNNNANNNN_ BspST5I y

SspD5I GGTGANNNNNNNNA - -

Sthl32I CCCGNNNNANNNN_ - -

StsI GGATGNNNNNNNNNNANNNN_ - -

TaqII GACCGANNNNNNNNN_NNA, CACCCANNNNNNNNN_NNA - -

Tthlllll CAARCANNNNNNNNN_NNA - -

UbaPI CGAACG - -

The notation is Λ means cut the upper strand and _ means cut the lower strand. If the upper and lower strand are cut at the same place, then only A appears .

Table 14 (FOKIact) 5 ' -cAcATccqTq TTgTT cAcqqATqTq-3 ' (VHEx881) 5'-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3' i note that VHEx881 is the reverse complement of the ON below ! [RC] 5’-cgCttcacTaag- ! Scab........ ! Synthetic 3-23 as in Table 206 i |TCT|AGA|gac|aacItct1aagIaatI act|etc|tacIttgIcag i atg|- ! Xbal... ! |aacIagCITTA|AGgI get|gag Igac|aCT|GCA|Gtc|tac|tat 11-3' I AfIII... (VHBA881) 51-cgCttcacTaag- ITCTIAGA|gac|aac|tctIaagIaatI act|ctc|tac|ttg|cag|atg|- ^ |aacIagCtTTA|AGgI get I gag IgacIaCTI GCA IGtcItac1 tat|tgt geg ag-3’ (VHBB881) 5'-cgCttcacTaag- |TCT|AGA|gac|aacItctIaagIaat|act|ctc|tac|ttg|cag|atg|-|aac|agCITTA|AGg|get|gag IgacIaCTI GCA IGtc|tac|tat|tgt Acg ag-3' (VH881PCR) 5'-cgCttcacTaagITCT|AGA|gac|aac -3'

Table 15: Use of Fokl as "Universal Restriction. Enzyme"

Fokl - for dsDNA, | represents sites of cleavage sites of cleavage 5'-cacGGATGtg—nnnnnnn|nnnnnnn-3'(SEQ ID NO:15) 3'-gtgCCTACac--nnnnnnnnnnnInnn-5'(SEQ ID N0:16)

RECOG NITion of Fokl

Case I 5'-...gtgItatt-actgtgc. . Substrate ....-3' (SEQ ID N0:17) 3'-cac-ataaItgacacq—i qtGTAGGcac\ 5’- caCATCCgtg/(SEQ ID NO:18)

Case II 5'-...gtgtattlagac-tgc..Substrate.. -.-3' (SEQ ID NO:19) rcacataa-tctqIacg-5' /gtgCCTACac \cacGGATGtg-3'(SEQ ID NO:20)

Case III (Case I rotated 180 degrees) /gtgCCTACac-5' \cacGGATGtq—| gtatcttIacaq-tcc-3' Adapter (SEQ ID NO:21) 3'-. . .cacagaa-tgtcIagg..substrate....-5' (SEQ ID NO:22)

Case IV (Case II rotated 180 degrees) 3'- gtGTAGGcacV (SEQ ID NO:23) j— caCATCCgtg/ 5'-aaaItctc-actaaqc

Substrate 3'-...ctc-agagItgactcg.. .-5' (SEQ ID NO:24)

Improved Fokl adapters

Fokl - for dsDNA, I represents sites of cleavage Case I

Stem 11, loop 5, stem 11, recognition 17 5'-...catgtgItatt-actgtgc.. Substrate....-3 ' 3'-otacac-ataaItgacacq—. rT—i gtGTAGGcacG T 5'- caCATCCgtgc C Ιχχΐ

Case II

Stem 10, loop 5, stem 10, recognition 18 5 ' -...gtgtattIagac-tgctgcc.. Substrate ....-3' ρΤη f—cacataa—tctqIacqacqq-5 ' T gtgCCTACac C fcacGGATGtg-3' ltxj

Case III (Case I rotated 180 degrees)

Stem 11, loop 5, stem 11, recognition 20 r Ti T TgtgCCTACac-5' G AcacGGATGtq—|

Ltt-1 atatctt I acaa-tccattctq-3 ' Adapter 3’-...cacagaa-tgtcIaggtaagac..substrate....-5'

Case IV (Case II rotated 180 degrees)

Stem 11, loop 4, stem 11, recognition 17 ΓΤΊ

3’- gtGTAGGcacc T [—caCATCCgtgg T S'-atcaaaItctc-actqaqc LTJ

Substrate 3 ' - . . .tagctc-agagItgactcg...-5'

BseRI I sites of cleavage 5 ' -cacGAGGAGnnnnnnnnnn I nnnnn-3 ' 3'-atactcctcnnnnnnnn|nnnnnnn-5'

RECOG

NITion of BseRI

Stem 11, loop 5, stem 11, recognition 19 3' -.......gaacat I cg-ttaagccagta.....5 ’ ΓΤ-ΤΊ cttgta-gcIaattcggtcat-3' C GCTGAGGAGTC--1

T cgactcctcag-5' An adapter for BseRI to cleave the substrate above. 1χ_I

Table 16 Human heavy chains bases 88.1 to 94.2

Number of sequences.......... 84 0

Number of Mismatchers......... Probe

Id Ntot 01234567 Name_Sequence............ Dot form............ 1 364 152 97 76 26 7 4 2 0 VHS881-1.1 gctgtgtattactgtgcgag gctgtgtattactgtgcgag 2 265 150 60 33 13 5 4 0 0 VHS881-1.2 gccgtgtattactgtgcgag ..c................. 3 96 14 34 16 10 5 7 9 1 VHS881-2.1 gccgtatattactgtgcgag ,.c..a.............. 4 20 03492200 VHS881-4.1 gccgtgtattactgtacgag ..c............a.... 5 95 25 36 18 11 2 2 0 1 VHS881-9.1 accatqtattactqtqcqaq .ca................ 840 341 230 147 69 21 19 11 2 341 571 718 787 808 827 838 840 88 89 90 91 92 93 94 95 Codon number as i.n Table 195

Recognition........... Stem...... Loop. Stem...... (VHS881-1.1) 5'-gctgtgtat|tact-gtgcgag cAcATccqTq TTqTT cAcqqATqTq-3' (VHS881-1.2) 5'-gccgtgtat1tact-gtgcgag cAcATccqTq TTgTT cAcqqATqTq-31 (VHS881-2.1) 5’-gccgtatatItact-gtgcgag cAcATccqTq TTqTT cAcqqATqTq-31 (VHS881-4.1) 5'-gccgtgtat|tact-gtacgag cAcATccqTq TTgTT cAcqqATqTq-3' (VHS881-9.1) 5'-qccatatatItact-qtqcqaq cAcATccqTq TTgTT cAcqqATqTg-3' | site of substrate cleavage (FOKIact) 5’-cAcATccgTgTTeTT cAcggATgTg-3' (VHEx881) 5'-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3' ! note that VHEx881 is the reverse complement of the ON below ! [RCJ 5’-cgCttcacTaag-I Scab........ ! Synthetic 3-23 as in Table 206 ! | TCT | AGA | gac | aac | tct | aag | aat | act | etc | tac | ttg | cag |atg | - ! Xbal... ! | aac | agC | TTA | AGg | get | gag | gac | aCT | GCA | Gtc | tac | tat 11-31 ! A fill... (VHBA881) 5'-cgCttcacTaag- | TCT | AGA | gac | aac | ret | aag | aat | act | etc | tac | ttg | cag | atg | -| aac | agC | ΤΓΑ | AGg | get | gag | gac | aCT | GCA | Gtc | tac | tat | tgt geg ag-3' (VHBB881) 5'-cgCttcacTaag- | TCT | AGA | gac | aac | tet | aag | aat | act | etc | tac | ttg | cag | atg | -| aac | agC | TTA | AGg | get | gag |gac | aCT | GCA | Gtc | tac | tat | tgt Aeg ag-3' (VH881PCR) 5'-cgCttcacTaag | TCT | AGA | gac | aac -3'

Table 17: Kappa, bases 12-30 ! I ID Ntot 0 1 2 3 4 5 6 Name Sequence........... Dot Form........... ! 1 84 40 21 20 1 2 0 0 SK12012 gacccagtctccatcctcc gacccagtctccatcctcc ! 2 32 19 3 6 2 1 0 1 SK12A17 gactcagtctccactctcc ...t.........ct.... | 3 26 17 8 1 0 0 0 0 SK12A27 gacgcagtctccaggcacc ...g.........gg.a.. 1 4 40 21 18 1 0 0 0 0 SKI2i\11 gacgcagtctccagccacc ...g.........g. a.. ! 182 97 50 28 3 3 0 1 ! 97 147 175 178 181 181 182 ] URE adapters: I Stem Loop. Stem Recognition........ (SzKB1230-O12) 5’-cAcATccgTg TTgTT cAcggATgTg ggAggATggAgAcTgggTc-3’ ! [RC] 5'-gacccagtctccatcctcc cAcATccgTg AAcAA cAcggATgTg-3' | Recognition........Stem loop. Stem I Fokl. Fokl. ! | Stem......Loop. Stem......Recognition........ (SzKB1230-A17) 5'-cAcATccgTg TTgTT cAcggATgTg ggAgAgTggAgAcTgAgTc-3' I [RC] 5'-gactcagtctccactctcc cAcATccgTg AAcAA cAcggATgTg-3' ! Recognition........Stem......loop. Stem...... | Fokl. Fold. ! | Stem......Loop. Stem......Recognition........ (SzKBl 230-A27) 5'-cAcATccgTg TTgTT cAcggATgTg ggTgccTggAgAcTgcgTc-3 ! [RC] 5'-gacgcagtctccaggcacc cAcATccgTg AAcAA cAcggATgTg-3' ! Recognition........Stem......loop. Stem...... | Fokl. Fokl. j 1 Stem......Loop. Stem......Recognition........ (SzKBl230-A11) 5’-cAcATccgTg TTgTT cAcggATgTg ggTggcTggAgAcTgcgTc-3 ! [RC] 5'-gacgcagtctccagccacc cAcATccgTg AAcAA cAcggATgTg-3' t Recognition........Stem......loop. Stem...... I Fokl. Fokl.

What happens in the upper strand: (SzKBl230-012*) 5'-gac cca gtc | tcc a-tc etc c-3’ t | Site of cleavage in substrate ! (SzKBl 230-A17*) 5'-gac tea gtc | tcc a-ct etc c-3' (SzKB1230-A27*) 5'-gac gca gtc| tcc a-gg cac c-3' (SzKB1230-Al 1 *) 5'-gac gca gtc | tcc a-gc cac c-3’ (kapextURE) 5'-ccTctactctTgTcAcAalasAcAA gAc ATc cAg-3’ !sense strand

Scab.............ApaLI. (kapextUREPCR) 5'-ccTctactctTgTcAcAgTg-3'

Scab............. (kaBROlUR) 5'“ggAggATggA cTggATgTcTTgTgcAcTgTgAcAAgAgTA gAgg-3 . R c f this one t [RC] 5--ccTctactctTgTcAcAaTacAcAA gAc ATc cAg tcc a-tc etc c-3 ON above is R.C. of th (kaBR02UR) 51 -ggAgAgTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3 above is R C of this one j (RCl 5 ' -ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-ct etc c-3 ON above is R.C. (kaBR03UR) 5' -ggTgccTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3' , is R C of this one ! [RC] 5'-ccTctactctTgTcAcAaIacAcAA gAc ATc cAg tcc a-gg cac c-3 ON above is R.C. (kaBR04UR) 5'-ggTggcTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3' is R C of this one ! [RC] 5 ’-ccTctactctTgTcAcAgTacAcAA gAc ATc cAg tcc a-gc cac c-3 ON above is R.C.

Scab.............ApaLI.

Table 18 Lambda URE adapters bases 13.3 to 19.3 I 1 Number of sequences.......... 128 1 1 Number of mismatches.............. I Id Ntot 0 1 2 3 4 16 7 8 Name_Sequence Dot form..... Γ 1 58 45 7 1 0 0 0 2 2 1 VL133-2a2 gtctcctggacagtcgatc gtctcctggacagtcgatc 12 16 10 1 0 1 0 1 1 0 2 VL133-31 ggccttgggacagacagtc .g.cttg a.ag.. !3 17 600041150 VL133-2c gtctcctggacagtcagtc ...............ag.. . j A in in 443742 VL133-lc ggccccagggcagagggtc .g.c..a..g...ag.g.. 1 128 64 8 11 5 8 5 11 11 5 | 64 72 83 88 96 101 112 123 128 ! I Stem loop. Stem Recognition........ (VL133-2a2) 5'-cAcATccgTg TTgTT cAcggATgTg gATcgAcTgTccAggAgAc-3 ! [RC] 5'-gtctcctggacagtcgatc cAcATccgTg AAcAA cAcggATgTg-3 | Recognition........Stem......Loop. Stem | 1 Stem......loop. Stem......Recognition........ (VL133-31) 5’-cAcATccgTg TTgTT cAcggATgTg gAcTgTcTgTcccAAggcc-3 i [RC] 5’-ggccttgggacagacagtc cAcATccgTg AAcAA cAcggATgTg 3 | Recognition........Stem Loop. Stem t j Stem......loop. Stem......Recognition........ (VO 33-2c) 5'-cAcATccgTg TTgTT cAcggATgTg gAcTgAcTgTccAggAgAc-3 [RC] 5'-gtctcctggacagtcagtc cAcATccgTg AAcAA cAcggATgTg- ! Recognition........Stem......Loop. Stem ! I Stem......loop. Stem......Recognition........ (VL133-lc) 5'-cAcATccgTg TTgTT cAcggATgTg gAcccTcTgcccTggggcc-3 [RC] 5'-ggccccagggcagagggtc cAcATccgTg AAcAA cAcggATg g-

What happens in the top strand: I | site of cleavage in the upper strand (VL133-2a2*) 5'-g tet cct g | ga cag teg ate t (VLl 33-31*) 5'-g gee ttg g | ga cag aca gtc ! (VLl 33-2c*) 5'-g tet cct g j ga cag tea gtc ! (VLl 33-lc*) 5'-g gee cca g | gg cag agg gtc i The following Extenders and Badges all encode the AA sequence of 2a2 for codons 1-15 I 1

(ON_LamExl33) 5’-ccTcTgAcTgAgT gcA cAg -I ! 2 3 4 5 6 7 8 9 10 11 12 AGt geT TtA acC caA ccG geT AGT gtT AGC ggT-t ! 13 14 15 teC ccG g! 2a2 I 1 (OINLLamBl-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg -t i 2 3 4 5 6 7 8 9 10 11 12 AGt geT TtA acC caA ccG geT AGT gtT AGC ggT- 1 ! 13 14 15 teC ccG g ga cag teg at-3'! 2a2 N.B. the actual seq is the i reverse complement of the i one shown, t (ON_LamB2-133) IRC] 5'-ccTcTgAcTgAgT gcA cAg -t ! 2 3 4 5 6 7 8 9 10 11 12 AGt geT TtA acC caA ccG geT AGT gtT AGC ggT-i ! 13 14 15 teC ccG g ga cag aca gt-3'! 31 7VLJ5. the actual seq is die t reverse complement of the t one shown. 1 (ON_LatnB3-l33) [RC] 5'-ccTcTgAcTgAgT gcA cAg - i t 2 3 4 5 6 7 8 9 10 11 12 AGt geT TtA acC caA ccG geT AGT gtT AGC ggT- 1 ! 13 14 15 teC ccG g ga cag tea gt -3'! 2c ALB. the actual seq is the I reverse complement of the I t one shown. !(ON_LamB4-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg - ; 2 3 4 5 6 7 8 9 10 11 12 AGt gcT TtA acC caA ccG gcT AGT gtT AGC ggT-s

I ! 13 14 15 tcC ccG g gg cag agg gt—3' ! lc N.B. the actual seg is the i reverse complement of the i one shown.

I (ON_Laml33PCR) 5'-ccTcTgAcTgAgT gcA cAg AGt gc-3'

Table 19: Cleavage of 75 human light chains.

En2vme Recognition*_Nch Ns Planned—location of—site

Afel AGCgct 0 0

Aflll Cttaag 0 0 HC FR3

AgeX Accggt 0 0

AscI GGcgcgcc 0 0 After LC

Bglll Agatct 0 0

BsiWI Cgtacg 0 0

BspDI ATcgat 0 0

BssHII Gcgcgc 0 0

BstBI TTcgaa 0 0

Dralll CACNNNgtg 0 0

EagI Cggccg 0 0

Fsel GGCCGGcc 0 0

FspI TGCgca 0 0

Hpal GTTaac 0 0

Mfel Caattg 0 0 HC FR1

Mlul Acgcgt 0 0

Ncol Ccatgg 0 0 Heavy chain signal

Nhel Gctagc 0 0 HC/anchor linker

jjotl GCggccgc 0 0 In linker after HC

Nrul TCGcga 0 0

PacI TTAATtaa 0 0

Pmel GTTTaaac 0 0

Pmll CACgtg 0 0

Pvul CGATcg 0 0

SacII CCGCgg 0 0

Sail Gtcgac 0 0

Sfil GGCCNNNNnggcc 0 0 Heavy Chain signal

SgfI GCGATcgc 0 0

SnaBI TACgta 0 0

StuI AGGcct 0 0

Xbal Tctaga 0 0 HC FR3

Aatll GACGTc 1 1

Acll AAcgtt 1 1

Asel ATtaat 1 1

BsmI GAATGCN 1 1

BspEI Tccgga 1 1 HC FR1

BstXI CCANNNNNntgg 1 1 HC FR2

DrdI GACNNNNnngtc 1 1

HindiII Aagctt 1 1

Pcil Acatgt 1 1

SapI gaagagc 1 1

Seal AGTact 1 1

SexAI Accwggt 1 1

Spel Actagt 1 1

Tlil Ctcgag 1 1

Xhol Ctcgag 1 1

Bcgl cgannnnnntgc 2 2

BlpI GCtnagc 2 2

BssSI Ctcgtg 2 2

BstAPI GCANNNNntgc 2 2

EspI GCtnagc 2 2

KasI Ggcgcc 2 2

PflMI CCANNNNntgg 2 2

XmnI GAANNnnttc 2 2

ApaLI Gtgcac 3 3 LC signal seq

Nael GCCggc 3 3

NgoMI Gccggc 3 3

PvuII CAGctg 3 3

RsrII CGgwccg 3 3

BsrBI GAGcgg 4 4

BsrDI GCAATGNNn 4 4

BstZ17I GTAtac 4 4

EcoRI Gaattc 4 4

SphI GCATGc 4 4

SspI AATatt 4 4

AccI GTmkac . 5 5

Bell Tgatca 5 5

BsmBI Nnnnnngagacg 5 5

BsrGI Tgtaca 5 5

Dral TTTaaa 6 6

Ndel CAtatg 6 6 HC FR4

Swal ATTTaaat 6 6

BamHI Ggatcc 7 7

SacI GAGCTc 7 7

BciVI GTATCCNNNNNN 8 8

BsaBI GATNNnnatc 8 8

Nsil ATGCAt 8 8

Bspl20I Gggccc 9 9 CHI

Apal GGGCCc 9 9 CHI

PspOOMI Gggccc 9 9

BspHI Tcatga 9 11

EcoRV GATatc 9 9

Ahdl GACNNNnngtc 11 11

Bbsl GAAGAC 11 14

Psil TTAtaa 12 12

Bsal GGTCTCNnnnn 13 15

Xmal Cccggg 13 14

Aval Cycgrg 14 16

Bgll GCCNNNNnggc 14 17

AlwNI CAGNNNctg 16 16

BspMI ACCTGC 17 19

Xcml CCANNNNNnnnntgg 17 26

BstEII Ggtnacc 19 22 HC FR4

Sse8387I CCTGCAgg 20 20

AvrII Cctagg' 22 22

Hindi GTYrac 22 22

Bsgl GTGCAG 27 29

MscI TGGcca 30 34

BseRI NNnnnnnnnnctcctc 32 35

Bsu36I CCtnagg 35 37

PstI CTGCAg 35 40

Ecil nnnnnnnnntccgcc 38 40

PpuMI RGgwccy 41 50

StyI Ccwwgg 44 73

EcoO109I RGgnccy 46 70

Acc65I Ggtacc 50 51

Kpnl GGTACc 50 51

Bpml ctccag 53 82

Avail Ggwcc 71 124 * cleavage occurs in the top strand after the last upper-case base. For REs that cut palindromic sequences, the lower strand is cut at the symmetrical site.

Table 20: Cleavage of 79 human heavy chains

Enzyme Recognition_Nch Ns Planned location of site

Af el AGCgct 0 0

Af III Cttaag 0 0 HC FR3

AscI GGcgcgcc 0 0 After LC

BsiWI Cgtacg 0 0

BspDI ATcgat 0 0

BssHII Gcgcgc 0 0

Fsel GGCCGGcc 0 0

Hpal GTTaac 0 0 _

Nhel Gctagc 0 0 HC Linker

Notl GCggccgc 0 0 In linker, HC/anchor

Nrul TCGcga 0 0

Nsil ATGCAt 0 0

PacI TTAATtaa 0 0

Pcil Acatgt 0 0

Pmel GTTTaaac 0 0

Pvul CGATcg 0 0

RsrII CGgwccg 0 0

SapI gaagagc 0 0

SfiX GGCCNNNNnggcc 0 0 HC signal seq

SgfI GCGATcgc 0 0

Swal ATTTaaat 0 0

Acll AAcgtt 1 1

Agel Accggt 1 1

Asel ATtaat 1 1

AvrII Cctagg 1 1

BsmI GAATGCN 1 1

BsrBI GAGcgg 1 1

BsrDI GCAATGNNn 1 1

Dral TTTaaa 1 1

FspI TGCgca 1 1

Hindlll Aagctt 1 1

Mfel Caattg 1 1 HC FR1

Nael GCCggc 1 1

NgoMI Gccggc 1 1

Spel Actagt 1 1

Acc65I Ggtacc 2 2

BstBI TTcgaa 2 2

Kpnl GGTACc 2 2

Mlul Acgcgt 2 2

Ncol Ccatgg 2 2 In HC signal seq

Ndel CAtatg 2 2 HC FR4

Pmll CACgtg 2 2

Xcml CCANNNNNnnnntgg 2 2

Bcgl cgannnnnntgc 3 3

Bell Tgatca 3 3

Bgll GCCNNNNnggc 3 3

BsaBI GATNNnnatc 3 3

BsrGI Tgtaca 3 3

SnaBI TACgta 3 3

Sse8387I CCTGCAgg 3 3

ApaLI Gtgcac 4 4 LC Signal/FR1

BspHI Tcatga 4 4

BssSI Ctcgtg 4 4

Psil TTAtaa 4 5

SphI GCATGc 4 4

Ahdl GACNNNnngtc 5 5

BspEI Tccgga 5 5 HC FR1

MscI TGGcca 5 5

SacI GAGCTc 5 5

Seal AGTact 5 5

SexAI Accwggt 5 6

SspI AATatt 5 5 ' Tlil Ctegag 5 5

Xhol Ctegag 5 5

Bbsl GAAGAC 7 8

BstAPI GCANNNNntgc 7 8

BstZ17I GTAtac 7 7

EcoRV GATatc 7 7

EcoRI Gaattc 8 8

BlpI GCtnagc 9 9

Bsu3 61 CCtnagg 9 9

Drain CACNNNgtg 9 9

EspI GCtnagc 9 9

StuI AGGcct 9 13

Xbal Tetaga 9 9 HC FR3

Bspl20I Gggccc 10 11 CHI

Apal GGGCCc 10 11 CHI

PspOOMI Gggccc 10 11

BciVI GTATCCNNNNNN 11 11

Sail Gtegae 11 12

DrdI GACNNNNnngtc 12 12

KasI Ggegee 12 12

Xmal Cccggg 12 14

Bglll Agatet 14 14

Hindi GTYrac 16 18

BamHI Ggatcc 17 17

PflMI CCANNNNntgg 17 18

BsmBI Nnnnnngagacg 18 21

BstXI CCANNNNNntgg 18 19 HC FR2

XmnI GAANNnnttc 18 18

SacII CCGCgg 19 19

PstI CTGCAg 20 24 '

PvuII CAGetg 20 22

Aval Cycgrg 21 24

EagI Cggccg 21 22

Aatll GACGTc 22 22

BspMI ACCTGC 27 33

AccI GTmkac 30 43

Styl Ccwwgg 36 49

AlwNI CAGNNNctg 38 44

Bsal GGTCTCNnnnn 38 44

PpuMI RGgwccy 43 46

Bsgl GTGCAG 44 54

BseRI NNnnnnnnnnctcctc 48 60

Ecil nnnnnnnnntccgcc 52 57

BstEII Ggtnacc 54 61 HC Fr4, 47/79 have one

Eco0109l RGgnccy 54 86

Bpml ctccag 60 121

Avail Ggwee 71 140

Table 21: MALIA3, annotated ! MALIA3 9532 bases ----------------------------------------------------------------------- 1 aat get act act att agt aga att gat gcc acc ttt tea get ege gee ! gene ii continued 49 cca aat gaa aat ata get aaa cag gtt att gac cat ttg ega aat gta 97 tet aat ggt caa act aaa tet act cgt teg cag aat tgg gaa tea act 145 gtt aca tgg aat gaa act tee aga cac cgt act tta gtt gca tat tta 193 aaa cat gtt gag eta cag cac cag att cag caa tta age tet aag cca 241 tee gca aaa.atg acc tet tat caa aag gag caa tta aag gta etc tet 289 aat cct gac ctg ttg gag ttt get tee ggt ctg gtt ege ttt gaa get 337 ega att aaa aeg ega tat ttg aag tet ttc ggg ett cct ett aat ett 385 ttt gat gca ate ege ttt get tet gac tat aat agt cag ggt aaa gac 433 ctg att ttt gat tta tgg tea ttc teg ttt tet gaa ctg ttt aaa gca 481 ttt gag ggg gat tea ATG aat att tat gac gat tee gca gta ttg gac ! RBS?...... Start gene x, ii continues 529 get ate cag tet aaa cat ttt act att acc ccc tet ggc aaa act tet 577 ttt gca aaa gcc tet ege tat ttt ggt ttt tat cgt cgt ctg gta aac 625 gag ggt tat gat agt gtt get ett act atg cct cgt aat tee ttt tgg 673 cgt tat gta tet gca tta gtt gaa tgt ggt att cct aaa tet caa ctg 721 atg aat ett tet acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att 769 aac gta gat ttt tet tee caa cgt cct gac tgg tat aat gag cca gtt

817 ett aaa ate gca TAA

! End X &amp; IX 832 ggtaattca ca i ! Ml E5 Q10 T15 843 ATG att aaa gtt gaa att aaa cca tet caa gcc caa ttt act act cgt

! Start gene V

I ! S17 S20 P25 E30 891 tet ggt gtt tet cgt cag ggc aag cct tat tea ctg aat gag cag ett l ! V35 E4 0 V45 939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ett gtc aag att act

I ! D50 A55 L60 987 ett gat gaa ggt cag cca gcc tat geg cct ggt cTG TAC Acc gtt cat I BsrGI... i L65 V70 S75 R80 1035 ctg tee tet ttc aaa gtt ggt cag ttc ggt tee ett atg att gac cgt

! P85 K87 end of V

1083 ctg ege etc gtt ccg get aag TAA C 1

1108 ATG gag cag gtc geg gat ttc gac aca att tat cag geg atg ! Start gene VII 1150 ata caa ate tee gtt gta ett tgt ttc geg ett ggt ata ate

I ! VII and IX overlap. ! ..... S2 V3 L4 V5 S10 1192 get ggg ggt caa agA TGA gt gtt tta gtg tat tet ttc gcc tet ttc gtt

! End VII

i I start IX ! L13 W15 G20 T25 E29 1242 tta ggt tgg tgc ett cgt agt ggc att aeg tat ttt acc cgt tta atg gaa

I 1293 act tcc tc ! ! ____ stop of IX, IX and VIII overlap by four bases 1301 ATG aaa aag tct tta gtc etc aaa gcc tet gta gee gtt get ace etc ! Start signal sequence of viii. i 1349 gtt ccg atg ctg tct ttc get get gag ggt gac gat ccc gca aaa geg ! mature VIII ---> 1397 gcc ttt aac tec ctg caa gcc tea geg acc gaa tat ate ggt tat geg 1445 tgg geg atg gtt gtt gtc att 1466 gtc ggc gca act ate ggt ate aag ctg ttt aag 1499 aaa ttc acc teg aaa gca ! 1515 ! ........... -35 . . t 1517 age tga taaaccgat acaattaaag gctccttttg ! ..... -10 . . . t 1552 gagccttttt ttttGGAGAt ttt ! S.D. underlined

I ! <------III signal sequence----------------------------->

! MKKLLFAIPLV 1575 caac GTG aaa aaa tta tta ttc gca att cct tta gtt ! 1611

I

! VPFYSHSAQ 1612 gtt cct ttc tat tct cac aGT gcA Cag tCT ! ApaLI...

I

' 1642 GTC GTG ACG CAG CCG CCC TCA GTG TCT GGG GCC CCA GGG CAG AGG GTC ACC ATC TCC TGC ACT GGG AGC AGC TCC AAC ATC GGG GCA ! BstEII...

1729 GGT TAT GAT GTA CAC TGG TAC CAG CAG CTT CCA GGA ACA GCC CCC AAA 1777 CTC CTC ATC TAT GGT AAC AGC AAT CGG CCC TCA GGG GTC CCT GAC CGA .

1825 TTC TCT GGC TCC AAG TCT GGC ACC TCA GCC TCC CTG GCC ATC ACT

1870 GGG CTC CAG GCT GAG GAT GAG GCT GAT TAT

1900 TAC TGC CAG TCC TAT GAC AGC AGC CTG AGT

. 1930 GGC CTT TAT GTC TTC GGA ACT GGG ACC AAG GTC ACC GTC ! BstEII...

1969 CTA GGT CAG CCC AAG GCC AAC CCC ACT GTC ACT

2002 CTG TTC CCG CCC TCC TCT GAG GAG CTC CAA GCC AAC AAG GCC ACA CTA

2050 GTG TGT CTG ATC AGT GAC TTC TAC CCG GGA GCT GTG ACA GTG GCC TGG

2098 AAG GCA GAT AGC AGC CCC GTC AAG GCG GGA GTG GAG ACC ACC ACA CCC

214 6 TCC AAA CAA AGC AAC AAC AAG TAC GCG GCC AGC AGC TAT CTG AGC CTG

2194 ACG CCT GAG CAG TGG AAG TCC CAC AGA AGC TAC AGC TGC CAG GTC ACG

2242 CAT GAA GGG AGC ACC GTG GAG AAG ACA GTG GCC CCT ACA GAA TGT TCA

2290 TAA TAA ACCG CCTCCACCGG GCGCGCCAAT TCTATTTCAA GGAGACAGTC ATA ! AscI.....

I ! PelB signal---------------------------------------------->

! MKYLL PTAAAGLLL L

2343 ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC i ! 16 17 18 19 20 21 22

! A A Q P A M A 2388 geG GCC cag ccG GCC_ato gee ! Sfil............. ! NgoMI...(1/2) ! Ncol.........

I i FR1(DP47/V3-23)--------------- I 23 24 25 26 27 28 29 30

I EVQLLESG

’ 2409 gaaIgttICAAITTGIttaI gag ItctIggtI

, | Mfel I i i --------------FR1-------------------------------------------- I 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

i GGLVQPGGSLRLSCA

2433 |ggclggt|cttIgtt i cagIcct|ggtIggtItctIttaIcgtIcttItctItgcI get I

I i ----pRl----------------> | . . . CDR1................I---FR2------ i 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 I asgftfssyamswvr ' 2 47 8 I get ITCC|GGA|ttcI act|ttcItctItCGITAC|Get|atg|tctItggIgttIcgC| ! | BspEI | I BsiWII IBstXI. j I -------FR2-------------------------------->1 · · · CDR2......... i 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 i qapgkglewvsaisg

2523 ICAaI get|ccT!GGtIaaa i ggt|ttgI gag ItggIgtt|tct|get I ate ItctIggtI ! ...BstXI I

I ! .....CDR2............................................I------ I 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

i SGGSTYYADSVKGRF

2 568 I tct|ggtIggcIagt|act I tac|tat|get IgacI tee IgttIaaaIggt|egeIttcI ! i i --------FR3-------------------------------------------------- i 9i 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! τ I SRDNSKNTLY LQM 2613 I act I ate|TCT|AGA|gacIaacItctIaagIaat|act|etc|tacIttgIcagIatg|

! | Xbal I j ! ----------------------------------------------------------->1 i 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! NSLRAEDTAVYYCAK

2658 i aacIagC|TTAIAGgI get I gag IgacIaCT|GCA IGtcItac|tat|tgc|get|aaaI ! |AfIII I I PstI I 1 ! .......CDR3.................I----FR4------------------------- ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

j DYEGTGYAFDIWGQG ’ 2703 I gacI tat Igaa|ggtI act Iggt I tat|get IttcIgaCI ΑΤΑ|TGgIggt|caa|ggt| I I Ndel I(1/4)

I I --------------FR4---------->1 ! 136 137 138 139 140 141 142

! Τ Μ V Τ V S S 274 8 | act|atG|GTCI ACC IgtcItctIagt

! | BstEII I ! From BstEII onwards, pV323 is same as pCESl, except as noted, i BstEII sites may occur in light chains; not likely to be unique in final ! vector. ; 143 144 145 146 147 148 149 150 151 152 i astkgpsvfp 27g9 qcc tcc acc aaG GGC CCa teg GTC TTC ccc , Bspl20I. BbsX. . . (2/2) I ApaI. ... t i 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 ; lapsskstsggtaal 2799 ctg gca ccC TCC TCc aag age acc tet ggg ggc aca geg gee ctg ! BseRI... (2/2) ! 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 i gclvkdyfpepvtvs 2844 ggc tgc ctg GTC AAG GAC TAC TTC CCc gaA CCG GTg aeg gtg teg i Agel.... i 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197

I WNSGALTS GVHT FPA 2889 tgg aac tea GGC GCC ctg acc age ggc gtc cac acc ttc ccg get ! KasI...(1/4) I 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212

! VLQSSGLYSLSSVVT 2934 gtc eta cag tCt age GGa etc tac tcc etc age age gta gtg acc i (Bsu36I...)(knocked out) i 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227

I VPSSSLGTQTYICNV 2979 gtg ccC tCt tet age tTG Ggc acc cag acc tac ate tgc aac gtg i (BstXI...........)N.B. destruction of BstXI &amp; Bpml sites. i 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 J nhkpsntkvdkkvep 3024 aat cac aag ccc age aac acc aag gtg gac aag aaa gtt gag ccc t ! 243 244 245

. KSCAAAHHHHHHSA 3069 aaa tet tgt GCG GCC GCt cat cac cac cat cat cac tet get i Notl...... i

i eQKLISEE DLNGAA 3111 gaa caa aaa etc ate tea gaa gag gat ctg aat ggt gee gca

I 1 i DINDDRM a s g a 3153 GAT ATC aac gat gat cgt atg get AGC ggc gee ! rEK cleavage site.......... Nhel... KasI... ! EcoRV..

J ! Domain 1 ----------------------------------------------------------- i aetvescla 3183 get gaa act gtt gaa agt tgt tta gca

I

I

I KPHTEISF 3210 aaa ccc cat aca gaa aat tea ttt

! TNVWKDDKT 3234 aCT AAC GTC TGG AAA GAC GAC AAA Act

I LDRYANYEGCLWNATGV 3261 tta gat cgt tac get aac tat gag ggt tgt ctg tgG AAT GCt aca ggc gtt i BsmI_ {

i VVCTGDETQCYGTWVPI ' 3312 gta gtt tgt act ggt GAC GAA ACT CAG TGT TAC GGT ACA TGG GTT cct att

I

! G L A I P E N 3363 ggg ett get ate cct gaa aat

I ! LI linker ------------------------------------

! EGGGSEGGGS 3384 gag ggt ggt ggc tet gag ggt ggc ggt tet

I

i EGGGSEGGGT 3414 gag ggt ggc ggt tet gag ggt ggc ggt act ! ! Domain 2 ------------------------------------ 3444 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat ate aac 3495 cct etc gac ggc act tat ccg cct ggt act gag caa aac ccc get aat cct 3546 aat cct tet ett GAG GAG tet cag cct ett aat act ttc atg ttt cag aat ! BseRI_ 3597 aat agg ttc ega aat agg cag ggg gca tta act gtt tat aeg ggc act 3645 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct

3693 gta tea tea aaa gee atg tat gac get tac tgg aac ggt aaa ttC AGA

I AlwNI

3741 GAC TGc get ttc cat tet ggc ttt aat gaa gat cca ttc gtt tgt gaa ! AlwNI 3789 tat caa ggc caa teg tet gac ctg cct caa cct cct gtc aat get ! 3834 ggc ggc ggc tet ! start L2 ------------------------------------------------------------- 3846 ggt ggt ggt tet 3858 ggt ggc ggc tet 3870 gag ggt ggt ggc tet gag ggt ggc ggt tet 3900 gag ggt ggc ggc tet gag gga ggc ggt tee 3930 ggt ggt ggc tet ggt ! end L2

I ! Domain 3 --------------------------------------------------------------

! SGDFDYEKMANANKGA 3945 tee ggt gat ttt gat tat gaa aag atg gca aac get aat aag ggg get

I

i MTENADENALQSDAKG 3993 atg acc gaa aat gee gat gaa aac geg eta cag tet gac get aaa ggc

I

I KLDSVATDYGAAIDGF 4041 aaa ett gat tet gtc get act gat tac ggt get get ate gat ggt ttc

I

! IGDVSGLANGNGATGD 4089 att ggt gac gtt tee ggc ett get aat ggt aat ggt get act ggt gat

I

! FAGSNSQMAQVGDGD N 4137 ttt get ggc tet aat tee caa atg get caa gtc ggt gac ggt gat aat

I ! splmnnfrqylpslpq 4185 tea cct tta atg aat aat ttc cgt caa tat tta cct tee etc cct caa

I SVECRP FVFSAGKPYE 4233 teg gtt gaa tgt ege cct ttt gtc ttt age get ggt aaa cca tat gaa

I | fsidcdkinlfr 4281 ttt tet att gat tgt gac aaa ata aac tta ttc cgt I End Domain 3 i GVFAFLLYVAT FMYV FI 4 0 4317 ggt gtc ttt geg ttt ett tta tat gtt gee acc ttt atg tat gta ttt ! start transmembrane segment

I

I S T F A N I L 4365 tet aeg ttt get aac ata ctg

I

! R N K E S 4386 cgt aat aag gag tet TAA ! stop of iii ! Intracellular anchor. t i Ml P2 V L L5 G I P L L10 L R F L G15

4404 tc ATG cca gtt ett ttg ggt att ccg tta tta ttg cgt ttc etc ggt ! Start VI

I 4451 ttc ett ctg gta act ttg ttc ggc tat ctg ett act ttt ett aaa aag 4499 ggc ttc ggt aag ata get att get att tea ttg ttt ett get ett att 4547 att ggg ett aac tea att ett gtg ggt tat etc tet gat att age get 4595 caa tta ccc tet gac ttt gtt cag ggt gtt cag tta att etc ccg tet 4643 aat geg ett ccc tgt ttt tat gtt att etc tet gta aag get get att 4691 ttc att ttt gac gtt aaa caa aaa ate gtt tet tat ttg gat tgg gat

I ! Ml A2 V3 F5 L10 G13

4739 aaa TAA t ATG get gtt tat ttt gta act ggc aaa tta ggc tet gga ! end VI Start gene I t ! 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

i KTLVSVGK1QD KIVA 4785 aag aeg etc gtt age gtt ggt aag att cag gat aaa att gta get

I i 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

! GCKIATNLDLRLQNL 4830 ggg tgc aaa ata gca act aat ett gat tta agg ett caa aac etc ! ! 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

! PQVGRFAKTPRVLRI 4875 ccg caa gtc ggg agg ttc get aaa aeg cct ege gtt ett aga ata

I ! 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

i PDKPSISDLLAIGRG 4920 ccg gat aag cct tet ata tet gat ttg ett get att ggg ege ggt

J ! 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

i NDSYDENKNGLLVLD 4965 aat gat tee tac gat gaa aat aaa aac ggc ttg ett gtt etc gat

I ! 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

i ECGTWFNTRSWNDKE 5010 gag tgc ggt act tgg ttt aat acc cgt tet tgg aat gat aag gaa

I ! 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

I RQPIIDWFLHARKLG 5055 aga cag ccg att att gat tgg ttt eta cat get cgt aaa tta gga ! 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

i WDIIFLVQDLSIVDK 5100 tgg gat att att ttt ett gtt cag gac tta tet att gtt gat aaa ! 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148

i QARSALAEHVVYCRR 5145 cag geg cgt tet gca tta get gaa cat gtt gtt tat tgt cgt cgt

I ' 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 i ldritlpfvgtlysl 5190 ctg gac aga att act tta cct ttt gtc ggt act tta tat tet ett i 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178

I ITGSKMPLPKLHVGV 5235 att act ggc teg aaa atg cct ctg cct aaa tta cat gtt ggc gtt ! 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193

j VKYGDSQLSPTVERW 5280 gtt aaa tat ggc gat tet caa tta age cct act gtt gag cgt tgg ! 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

i LYTGKNLYNAYDTKQ 5325 ett tat act ggt aag aat ttg tat aac gca tat gat act aaa cag i 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 I afssnydsgvysylt 5370 get ttt tet agt aat tat gat tee ggt gtt tat tet tat tta aeg ' 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

i PYLSHGRYFKPLNLG 5415 cct tat tta tea cac ggt egg tat ttc aaa cca tta aat tta ggt i 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 i qkmkltkiylkkfsr 5460 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tet ege ' 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268

i VLCLAIGFASAFTYS 5505 gtt ett tgt ett geg att gga ttt gca tea gca ttt aca tat agt i 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283

i YITQPKPEVKKVVSQ 5550 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tet cag i 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298

i TYDFDKFTIDSSQRL 5595 acc tat gat ttt gat aaa ttc act att gac tet tet cag cgt ett

I i 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 i nlsyryvfkdskgkl

5640 aat eta age tat ege tat gtt ttc aag gat tet aag gga aaa TTA , PacI

I ! 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 ! insddlqkqgyslty

5685 ATT AAt age gac gat tta cag aag caa ggt tat tea etc aca tat ! PacI

I ! 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343

! ilDLCTVS IKKGNSNE

! iv Ml K

5730 att gat tta tgt act gtt tee att aaa aaa ggt aat tea aAT Gaa ! Start IV i ! 344 345 346 347 348 349

! i I V K C N .End of I ! iv L3 L N5 V 17 N F V10 5775 att gtt aaa tgt aat TAA T TTT GTT ! IV continued..... 5800 ttc ttg atg ttt gtt tea tea tet tet ttt get cag gta att gaa atg 5848 aat aat teg cct ctg ege gat ttt gta act tgg tat tea aag caa tea 5896 ggc gaa tee gtt att gtt tet ccc gat gta aaa ggt act gtt act gta 5944 tat tea tet gac gtt aaa cct gaa aat eta ege aat ttc ttt att tet 5992 gtt tta cgt get aat aat ttt gat atg gtt ggt tea att cct tee ata 6040 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca 6088 tea tet gat aat cag gaa tat gat gat aat tee get cct tet ggt ggt 6136 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat 6184 aac gtt egg gca aag gat tta ata ega gtt gtc gaa ttg ttt gta aag 6232 tet aat act tet aaa tee tea aat gta tta tet att gac ggc tet aat 6280 eta tta gtt gtt TCT gca cct aaa gat att tta gat aac ett cct caa ! ApaLI removed 6328 ttc ett tet act gtt gat ttg cca act gac cag ata ttg att gag ggt 6376 ttg ata ttt gag gtt cag caa ggt gat get tta gat ttt tea ttt get 6424 get ggc tet cag cgt ggc act gtt gca ggc ggt gtt aat act gac ege 6472 etc acc tet gtt tta tet tet get ggt ggt teg ttc ggt att ttt aat 6520 ggc gat gtt tta ggg eta tea gtt ege gca tta aag act aat age cat 6568 tea aaa ata ttg tet gtg cca cgt att ett aeg ett tea ggt cag aag 6616 ggt tet ate tet gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg ! MscI_ 6664 act ggt gaa tet gee aat gta aat aat cca ttt cag aeg att gag cgt 6712 caa aat gta ggt att tee atg age gtt ttt cct gtt gca atg get ggc 67 60 ggt aat att gtt ctg gat att acc age aag gee gat agt ttg agt tet 6808 tet act cag gca agt gat gtt att act aat caa aga agt att get aca 6856 aeg gtt aat ttg cgt gat gga cag act ett tta etc ggt ggc etc act 6904 gat tat aaa aac act tet caa gat tet ggc gta ccg ttc ctg tet aaa 6952 ate cct tta ate ggc etc ctg ttt age tee ege tet gat tee aac gag 7000 gaa age aeg tta tac gtg etc gtc aaa gca acc ata gta ege gee ctg

7048 TAG cggcgcatt ! End IV 7060 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 7120 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcGCCGGCt ttccccgtca ! NgoMI_ 7180 agctctaaat cgggggctcc ctttagggtt ccgatttagt getttaegge acctcgaccc 7240 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt ! Drain_ 7300 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac ! DrdI_ 7360 aacactcaac cctatctcgg getattettt tgatttataa gggattttgc cgatttcgga 7420 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 7480 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga ! PvuII. BsmBI. 7540 aaaaccaccc tGGATCC AAGCTT ! BamHI Hindlll (½) 1 Insert carrying bla gene 7563 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc

7600 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct , BciVI 7660 gataaatgct tcaataatat tgaaaaAGGA AGAgt j RBS.?... ! Start bla gene 7695 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt 7746 tgc ctt cct gtt ttt get cac cca gaa aeg ctg gtg aaa gta aaa gat get 7797 gaa gat cag ttg ggC gCA CGA Gtg ggt tac ate gaa ctg gat etc aac age ! BssSI... i ApaLI removed 7848 ggt aag ate ctt gag agt ttt ege ccc gaa gaa cgt ttt cca atg atg age 7899 act ttt aaa gtt ctg eta tgt cat aca eta tta tee cgt att gac gcc ggg

7950 caa gaG CAA CTC GGT CGc egg gcg egg tat tet cag aat gac ttg gtt gAG ! Bcql _ Seal 8001 TAC Tea cca gtc aca gaa aag cat ctt aeg gat ggc atg aca gta aga gaa ! Scal_ 8052 tta tgc agt get gcc ata ace atg agt gat aac act gcg gcc aac tta ctt 8103 ctg aca aCG ATC Gga gga ccg aag gag eta acc get ttt ttg cac aac atg j Pvul_ 8154 ggg gat cat gta act ege ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc 8205 ata cca aac gac gag cgt gac acc aeg atg cct gta gca atg cca aca aeg 8256 tTG CGC Aaa eta tta act ggc gaa eta ctt act eta get tee egg caa caa ! FspI.... | 8307 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg ege teg 8358 GCC ctt ccG GCt ggc tgg ttt att get gat aaa tet gga gcc ggt gag cgt ! Bgll_ 8409 gGG TCT Cgc ggt ate att gca gca ctg ggg cca gat ggt aag ccc tee cgt ! Bsal_ 8460 ate gta gtt ate tac aeG ACg ggg aGT Cag gca act atg gat gaa ega aat i Ahdl_ 8511 aga cag ate get gag ata ggt gcc tea ctg att aag cat tgg TAA ctgt i stop 8560 cagaccaagt ttactcatat ataetttaga ttgatttaaa acttcatttt taatttaaaa 8620 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 8680 cgttccactg taegtaagae cccc 8704 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct ! Hindlll Sail.. ! (2/2) Hindi 8740 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgegatett

I

8790 CCTGAGG ! Bsu36I_ 8797 ccgat actgtcgtcg tcccctcaaa ctggcagatg 8832 cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg 8892 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc 8952 tggetacagg aaggccagac gegaattatt tttgatggcg ttcctattgg ttaaaaaatg

9012 aqctqattta acaaaaattt aaegegaatt ttaacaaaat attaacgttt acaATTTAAA I Swal. .. 9072 Tatttgetta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat , RBS?

9131 ATG att gac atg eta gtt tta ega tta ccg ttc ate gat tet ctt gtt tgc ! Start gene II 9182 tee aga etc tea ggc aat gac ctg ata gcc ttt gtA GAT CTc tea aaa ata i Bglll... 9233 get acc etc tee ggc atg aat tta tea get aga aeg gtt gaa tat cat att 9284 gat ggt gat ttg act gtc tcc ggc ctt tct cac cct ttt gaa tct tta cct 9335 aca cat tac tea ggc att gca ttt aaa ata tat gag ggt tct aaa aat ttt 9386 tat cct tgc gtt gaa ata aag get tct ccc gca aaa gta tta cag ggt cat 9437 aat gtt ttt ggt aca acc gat tta get tta tgc tct gag get tta ttg ctt 9488 aat ttt get aat tct ttg cct tgc ctg tat gat tta ttg gat gtt ! 9532 ! gene II continues

Table 21B: Sequence of MALIA3, condensed

LOCUS MALIA3 95 32 CIRCULAR

ORIGIN

1 AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT

61 ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT

12i CGTTCGCAGA ATTGGGAATC AACTGTTACA TGGAATGAAA CTTCCAGACA CCGTACTTTA

181 GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA

241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG

361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT

4 21 CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA

481 TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT

54x AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT

601 GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT

661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG

721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT

781 TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA

841 CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT

901 CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG

961 AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC

1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC

1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT

1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT

1201 CAAAGATGAG TGTTTTAGTG TATTCTTTCG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA

1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT

1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA

1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA

1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA

1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT

1561 TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC

1621 TATTCTCACA GTGCACAGTC TGTCGTGACG CAGCCGCCCT CAGTGTCTGG GGCCCCAGGG

1 gg i CAGAGGGTCA CCATCTCCTG CACTGGGAGC AGCTCCAACA TCGGGGCAGG TTATGATGTA

1741 CACTGGTACC AGCAGCTTCC AGGAACAGCC CCCAAACTCC TCATCTATGG TAACAGCAAT

1801 CGGCCCTCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT CTGGCACCTC AGCCTCCCTG

1861 GCCATCACTG GGCTCCAGGC TGAGGATGAG GCTGATTATT ACTGCCAGTC CTATGACAGC

1921 AGCCTGAGTG GCCTTTATGT CTTCGGAACT GGGACCAAGG TCACCGTCCT AGGTCAGCCC

1981 AAGGCCAACC CCACTGTCAC TCTGTTCCCG CCCTCCTCTG AGGAGCTCCA AGCCAACAAG

2041 GCCACACTAG TGTGTCTGAT CAGTGACTTC TACCCGGGAG CTGTGACAGT GGCCTGGAAG

2101 GCAGATAGCA GCCCCGTCAA GGCGGGAGTG GAGACCACCA CACCCTCCAA ACAAAGCAAC

2161 AACAAGTACG CGGCCAGCAG CTATCTGAGC CTGACGCCTG AGCAGTGGAA GTCCCACAGA

2221 AGCTACAGCT GCCAGGTCAC GCATGAAGGG AGCACCGTGG AGAAGACAGT GGCCCCTACA

2281 GAATGTTCAT AATAAACCGC CTCCACCGGG CGCGCCAATT CTATTTCAAG GAGACAGTCA

2341 TAATGAAATA CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCG GCCCAGCCGG

2401 CCATGGCCGA AGTTCAATTG TTAGAGTCTG GTGGCGGTCT TGTTCAGCCT GGTGGTTCTT

2461 TACGTCTTTC TTGCGCTGCT TCCGGATTCA CTTTCTCTTC GTACGCTATG TCTTGGGTTC

2521 GCCAAGCTCC TGGTAAAGGT TTGGAGTGGG TTTCTGCTAT CTCTGGTTCT GGTGGCAGTA

2581 CTTACTATGC TGACTCCGTT AAAGGTCGCT TCACTATCTC TAGAGACAAC TCTAAGAATA

2641 CTCTCTACTT GCAGATGAAC AGCTTAAGGG CTGAGGACAC TGCAGTCTAC TATTGCGCTA

2701 AAGACTATGA AGGTACTGGT TATGCTTTCG ACATATGGGG TCAAGGTACT ATGGTCACCG

2761 TCTCTAGTGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC TCCAAGAGCA

2821 CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC GAACCGGTGA

2881 CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTCCA CACCTTCCCG GCTGTCCTAC

2941 AGTCTAGCGG ACTCTACTCC CTCAGCAGCG TAGTGACCGT GCCCTCTTCT AGCTTGGGCA

3001 CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG GACAAGAAAG

3061 TTGAGCCCAA ATCTTGTGCG GCCGCTCATC ACCACCATCA TCACTCTGCT GAACAAAAAC

3121 TCATCTCAGA AGAGGATCTG AATGGTGCCG CAGATATCAA CGATGATCGT ATGGCTGGCG

3181 CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG

3241 TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGTTGT CTGTGGAATG

3301 CTACAGGCGT TGTAGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA TGGGTTCCTA

3361 TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGGTGGCGGT TCTGAGGGTG

3421 GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT

3481 ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA

3541 ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA

3601 GGTTCCGAAA TAGGCAGGGG GCATTAACTG TTTATACGGG CACTGTTACT CAAGGCACTG

3661 ACCCCGTTAA AACTTATTAC CAGTACACTC CTGTATCATC AAAAGCCATG TATGACGCTT

3721 ACTGGAACGG TAAATTCAGA GACTGCGCTT TCCATTCTGG CTTTAATGAA GATCCATTCG

3781 TTTGTGAATA TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG

3841 GCTCTGGTGG TGGTTCTGGT GGCGGCTCTG AGGGTGGTGG CTCTGAGGGT GGCGGTTCTG

3901 AGGGTGGCGG CTCTGAGGGA GGCGGTTCCG GTGGTGGCTC TGGTTCCGGT GATTTTGATT

3961 ATGAAAAGAT GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC

4021 TACAGTCTGA CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG

4081 ATGGTTTCAT TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG

4141 CTGGCTCTAA TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA

4201 ATTTCCGTCA ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTA

4261 GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG

4321 TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA

4381 TACTGCGTAA TAAGGAGTCT TAATCATGCC AGTTCTTTTG GGTATTCCGT TATTATTGCG

4441 TTTCCTCGGT TTCCTTCTGG TAACTTTGTT CGGCTATCTG CTTACTTTTC TTAAAAAGGG

4501 CTTCGGTAAG ATAGCTATTG CTATTTCATT GTTTCTTGCT CTTATTATTG GGCTTAACTC

4561 AATTCTTGTG GGTTATCTCT CTGATATTAG CGCTCAATTA CCCTCTGACT TTGTTCAGGG

4621 TGTTCAGTTA ATTCTCCCGT CTAATGCGCT TCCCTGTTTT TATGTTATTC TCTCTGTAAA

4681 GGCTGCTATT TTCATTTTTG ACGTTAAACA AAAAATCGTT TCTTATTTGG ATTGGGATAA

4741 ATAATATGGC TGTTTATTTT GTAACTGGCA AATTAGGCTC TGGAAAGACG CTCGTTAGCG

4801 TTGGTAAGAT TCAGGATAAA ATTGTAGCTG GGTGCAAAAT AGCAACTAAT CTTGATTTAA

4861 GGCTTCAAAA CCTCCCGCAA GTCGGGAGGT TCGCTAAAAC GCCTCGCGTT CTTAGAATAC

4921 CGGATAAGCC TTCTATATCT GATTTGCTTG CTATTGGGCG CGGTAATGAT TCCTACGATG

4981 AAAATAAAAA CGGCTTGCTT GTTCTCGATG AGTGCGGTAC TTGGTTTAAT ACCCGTTCTT

5041 GGAATGATAA GGAAAGACAG CCGATTATTG ATTGGTTTCT ACATGCTCGT AAATTAGGAT

5101 GGGATATTAT TTTTCTTGTT CAGGACTTAT CTATTGTTGA TAAACAGGCG CGTTCTGCAT

5161 TAGCTGAACA TGTTGTTTAT TGTCGTCGTC TGGACAGAAT TACTTTACCT TTTGTCGGTA

5221 CTTTATATTC TCTTATTACT GGCTCGAAAA TGCCTCTGCC TAAATTACAT GTTGGCGTTG

5281 TTAAATATGG CGATTCTCAA TTAAGCCCTA CTGTTGAGCG TTGGCTTTAT ACTGGTAAGA

5341 ATTTGTATAA CGCATATGAT ACTAAACAGG CTTTTTCTAG TAATTATGAT TCCGGTGTTT

5401 ATTCTTATTT AACGCCTTAT TTATCACACG GTCGGTATTT CAAACCATTA AATTTAGGTC

5461 AGAAGATGAA ATTAACTAAA ATATATTTGA AAAAGTTTTC TCGCGTTCTT TGTCTTGCGA

5521 TTGGATTTGC ATCAGCATTT ACATATAGTT ATATAACCCA ACCTAAGCCG GAGGTTAAAA

5581 AGGTAGTCTC TCAGACCTAT GATTTTGATA AATTCACTAT TGACTCTTCT CAGCGTCTTA

5641 ATCTAAGCTA TCGCTATGTT TTCAAGGATT CTAAGGGAAA ATTAATTAAT AGCGACGATT

5701 TACAGAAGCA AGGTTATTCA CTCACATATA TTGATTTATG TACTGTTTCC ATTAAAAAAG

5761 GTAATTCAAA TGAAATTGTT AAATGTAATT AATTTTGTTT TCTTGATGTT TGTTTCATCA

5821 TCTTCTTTTG CTCAGGTAAT TGAAATGAAT AATTCGCCTC TGCGCGATTT TGTAACTTGG

5881 TATTCAAAGC AATCAGGCGA ATCCGTTATT GTTTCTCCCG ATGTAAAAGG TACTGTTACT

5941 GTATATTCAT CTGACGTTAA ACCTGAAAAT CTACGCAATT TCTTTATTTC TGTTTTACGT

6001 GCTAATAATT TTGATATGGT TGGTTCAATT CCTTCCATAA TTCAGAAGTA TAATCCAAAC

6061 AATCAGGATT ATATTGATGA ATTGCCATCA TCTGATAATC AGGAATATGA TGATAATTCC

6121 GCTCCTTCTG GTGGTTTCTT TGTTCCGCAA AATGATAATG TTACTCAAAC TTTTAAAATT

6181 AATAACGTTC GGGCAAAGGA TTTAATACGA GTTGTCGAAT TGTTTGTAAA GTCTAATACT

6241 TCTAAATCCT CAAATGTATT ATCTATTGAC GGCTCTAATC TATTAGTTGT TTCTGCACCT

6301 AAAGATATTT TAGATAACCT TCCTCAATTC CTTTCTACTG TTGATTTGCC AACTGACCAG

6361 ATATTGATTG AGGGTTTGAT ATTTGAGGTT CAGCAAGGTG ATGCTTTAGA TTTTTCATTT

6421 GCTGCTGGCT CTCAGCGTGG CACTGTTGCA GGCGGTGTTA ATACTGACCG CCTCACCTCT

6481 GTTTTATCTT CTGCTGGTGG TTCGTTCGGT ATTTTTAATG GCGATGTTTT AGGGCTATCA

6541 GTTCGCGCAT TAAAGACTAA TAGCCATTCA AAAATATTGT CTGTGCCACG TATTCTTACG

6601 CTTTCAGGTC AGAAGGGTTC TATCTCTGTT GGCCAGAATG TCCCTTTTAT TACTGGTCGT

6661 GTGACTGGTG AATCTGCCAA TGTAAATAAT CCATTTCAGA CGATTGAGCG TCAAAATGTA

6721 GGTATTTCCA TGAGCGTTTT TCCTGTTGCA ATGGCTGGCG GTAATATTGT TCTGGATATT

6781 ACCAGCAAGG CCGATAGTTT GAGTTCTTCT ACTCAGGCAA GTGATGTTAT TACTAATCAA 6841 AGAAGTATTG CTACAACGGT TAATTTGCGT GATGGACAGA CTCTTTTACT CGGTGGCCTC 6901 ACTGATTATA AAAACACTTC TCAAGATTCT GGCGTACCGT TCCTGTCTAA AATCCCTTTA 6961 ATCGGCCTCC TGTTTAGCTC CCGCTCTGAT TCCAACGAGG AAAGCACGTT ATACGTGCTC 7021 GTCAAAGCAA CCATAGTACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 7081 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT 7141 CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC 7201 TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTTGGGTGA 7261 TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC 7321 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGG 7381 CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGAA CCACCATCAA ACAGGATTTT 7441 CGCCTGCTGG GGCAAACCAG CGTGGACCGC TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG 7501 AAGGGCAATC AGCTGTTGCC CGTCTCACTG GTGAAAAGAA AAACCACCCT GGATCCAAGC 7561 TTGCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA

7 621 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT 7681 GAAAAAGGAA GAGTATGAGT attcaacatt tccgtgtcgc CCTTATTCCC TTTTTTGCGG 7741 CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA GATGCTGAAG 7801 ' ATCAGTTGGG CGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG 7861 AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTC 7921 ATACACTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGG GCGCGGTATT 7981 CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA 8041 CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG GCCAACTTAC 8101 TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGGATC 8161 ' ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC 8221 GTGACACCAC GATGCCTGTA GCAATGCCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC 8281 TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT AAAGTTGCAG 8341 GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG 8401 GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG CCCTCCCGTA 84 61 TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG 8521 CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT TACTCATATA 8581 TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG AAGATCCTTT 8641 TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGT ACGTAAGACC 8701 CCCAAGCTTG TCGACTGAAT GGCGAATGGC GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG 8761 TGCCGGAAAG CTGGCTGGAG TGCGATCTTC CTGAGGCCGA TACTGTCGTC GTCCCCTCAA

8 821 ACTGGCAGAT GCACGGTTAC GATGCGCCCA TCTACACCAA CGTAACCTAT CCCATTACGG 8881 TCAATCCGCC GTTTGTTCCC ACGGAGAATC CGACGGGTTG TTACTCGCTC ACATTTAATG 8941 TTGATGAAAG CTGGCTACAG GAAGGCCAGA CGCGAATTAT TTTTGATGGC GTTCCTATTG 9001 GTTAAAAAAT GAGCTGATTT AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGTT 9061 TACAATTTAA ATATTTGCTT ATACAATCTT CCTGTTTTTG GGGCTTTTCT GATTATCAAC 9121 CGGGGTACAT ATGATTGACA TGCTAGTTTT ACGATTACCG TTCATCGATT CTCTTGTTTG 9181 CTCCAGACTC TCAGGCAATG ACCTGATAGC CTTTGTAGAT CTCTCAAAAA TAGCTACCCT 9241 CTCCGGCATG AATTTATCAG CTAGAACGGT TGAATATCAT ATTGATGGTG ATTTGACTGT 9301 CTCCGGCCTT TCTCACCCTT TTGAATCTTT ACCTACACAT TACTCAGGCA TTGCATTTAA 9361 AATATATGAG GGTTCTAAAA ATTTTTATCC TTGCGTTGAA ATAAAGGCTT CTCCCGCAAA 9421 AGTATTACAG GGTCATAATG TTTTTGGTAC AACCGATTTA GCTTTATGCT CTGAGGCTTT 9481 ATTGCTTAAT TTTGCTAATT CTTTGCCTTG CCTGTATGAT TTATTGGATG TT

Table 22: Primers used in RACE amplification:

Heavy chain

HuCU-EOR (1st PCR) 5'-TGG AAG AGG CAC GTT CTT TTC TTT-31

HuCp-Nested (2nd PCR) 5’ CTT TTC TTT GTT GCC GTT GGG GTG-31

Kappa light chain

HuCkFor (1st PCR) 5'-ACA CTC TCC CCT GTT GAA GCT CTT-3'

HuCkForAscI(2nd PCR) 5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA ACA CTC TCC CCT GTT GAA GCT CTT-3'

Lambda light chain HuClambdaFor (1st PCR)

HuCL2-F0R 5'-TGA ACA TTC TGT AGG GGC CAC TG-3'

HuCL7-FOR 5'-AGA GCA TTC TGC AGG GGC CAC TG-3’

HuClambdaForAscI (2nd PCR)

HUCL2-FOR-ASC 5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA TGA ACA TTC TGT AGG GGC CAC TG-3'

HUCL7-FOR-ASC 5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA AGA GCA TTC TGC AGG GGC CAC TG-3'

GeneRAcer 5' Primers provided with the kit (Invitrogen) 5'A 1st PCR 5'CGACTGGAGCACGAGGACACTGA 3' 5’NA 2nd pCR 5'GGACACTGACATGGACTGAAGGAGTA-3'

Table 23: ONs used in Capture of kappa light chains using CJ method and BsmM All ONs are written 5' to 3'. REdapters (6) ON_20SK15012 gggAggATggAgAcTgggTc ON_20SK15L12 gggAAgATggAgAcTgggTc ON_20SK15A17 gggAgAgTggAgAcTgAgTc ON_20SK15A27 gggTgccTggAgAcTgcgTc 0N_2 0SK15A11 gggTggcTggAgAcTgcgTc ON_20SK15B3 gggAgTcTggAgAcTgggTc

Bridges (6) kapbril012 gggAggATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg kapbri1L12 gggAAgATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg kapbrilA17 gggAgAgTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg kapbri1A27 gggTgccTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg kapbri1A11 gggTggcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg kapbri1B3 gggAgTcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg

Extender (5’ biotinylated) kapextlbio ccTcTgTcAcAgTgcAcAAgAcATccAgATgAcccAgTcTcc

Primers kaPCRtl ccTcTgTcAcAgTgcAcAAgAc kapfor 5'-aca etc tcc cct gtt gaa get ctt-3'

Table 24: PCR program for amplification of kappa DNA 95°C 5 minutes 95°C 15 seconds 65°C 30 seconds 72°C 1 minute 72°C 7 minutes 4°C hold

Reagents (100 ul reaction):

Template 50 ng lOx turbo PCR buffer lx

turbo Pfu 4U (JNTPs 200 μΜ each

kaPCRtl 300 nM

kapfor 300 nM

Table 25: h3401-h2 captured Via CJ with BsmAI ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 iSAQDIQMTQSPATLS aGTGCACaa gac ate cag atg acc cag tet cca gee ace ctg tet ! ApaLI... a gee acc ! L25,L6,L20,L2,L16,A11 ! Extender.................................Bridge... ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 IVSPGERATLSCRASQ gtg tet cca ggg gaa agg gee acc etc tee tgc agg gee agt cag ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 1SVSNN LAWYQQKPGQ agt gtt agt aac aac tta gee tgg tac cag cag aaa cct ggc cag ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1VPRLLIYGASTRATD gtt ccc agg etc etc ate tat ggt gca tee acc agg gee act gat ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 1IPARFSGSGSGTDFT ate cca gee agg ttc agt ggc agt ggg tet ggg aca gac ttc act ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 1LTISRLEPEDFAVYY etc acc ate age aga ctg gag cct gaa gat ttt gca gtg tat tac ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 1CQRYGSSPGWTFGQG tgt cag egg tat ggt age tea ccg ggg tgg aeg ttc ggc caa ggg ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 1TKVEI KRTV AAPSVF acc aag gtg gaa ate aaa ega act gtg get gca cca tet gtc ttc ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 MFPPSDEQLK.SGTAS ate ttc ccg cca tet gat gag cag ttg aaa tet gga act gee tet ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 1VVCLLNNFYPREAKV gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gee aaa gta ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 1QWKVDNALQSGNSQE cag tgg aag gtg gat aac gee etc caa teg ggt aac tee cag gag ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 1SVTEQDSKDSTYSLS agt gtc aca gag cag gac age aag gac age acc tac age etc age ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

iSTLTLSKADYEKHKV age ace ctg aeg ctg age aaa gca gac tac gag aaa cac aaa gtc i 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 i yacevthqglsspvt ’ tac gee tgc gaa gtc ace cat cag ggc ctg age teg cct gtc aca i 211 212 213 214 215 216 217 218 219 220 221 222 223

iKSFNKGECKGEFA aag age ttc aac aaa gga gag tgt aag ggc gaa ttc gc.....

Table 26: h3401-d8 KAPPA captured with CJ and Bsmkl ! 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 1SAQDIQMTQSPATLS aGT GCA Caa gac ate cag atg acc cag tet cct gee acc ctg tet

! ApaLI...Extender.........................g gee acc ! L25,L6,L20,L2,L16,AH ! A GCC ACC CTG TCT! L2 ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1VSPGERATLSCRASQ gtg tet cca ggt gaa aga gee acc etc tee tgc agg gee agt cag ! GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L2 ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 1NLLSNLAWYQQKPGQ aat ett etc age aac tta gee tgg tac cag cag aaa cct ggc cag ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1APRLLIYGASTGAIG get ccc agg etc etc ate tat ggt get tee acc ggg gee att ggt ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 1IPARFSGSGSGTEFT ate cca gee agg ttc agt ggc agt ggg tet ggg aca gag ttc act ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ILTISSLQSEDFAVYF etc acc ate age age ctg cag tet gaa gat ttt gca gtg tat ttc ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 1CQQYGTSPPTFGGGT tgt cag cag tat ggt acc tea ccg ccc act ttc ggc gga ggg acc ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 1KVEIKRTVAAPSVFI aag gtg gag ate aaa ega act gtg get gca cca tet gtc ttc ate ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 1FPPSDEQLKSGTASV ttc ccg cca tet gat gag cag ttg aaa tet gga act gee tet gtt ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 1VCPLNNFYPREAKVQ gtg tgc ccg ctg aat aac ttc tat ccc aga gag gee aaa gta cag ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 1WKVDNALQSGNSQES tgg aag gtg gat aac gee etc caa teg ggt aac tee cag gag agt ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 1VTEQDNKDSTYSLSS gtc aca gag cag gac aac aag gac age acc tac age etc age age ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 1TLTLSKVDYEKHEVY acc ctg acg ctg age aaa gta gac tac gag aaa cac gaa gtc tac ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 IACEVTHQGLSSPVTK gee tgc gaa gtc acc cat cag ggc ett age teg ccc gtc acg aag ! 211 212 213 214 215 216 217 218 219 220 221 222 223 ISFNRGECKKEFV age ttc aac agg gga gag tgt aag aaa gaa ttc gtt t

Table 27: V3-23 VH framework with variegated codons shown ! ! 17 18 19 20 21 22

! A Q P A M A 5'-ctg tct gaa cG GCC cag ccG GCC atg gcc 29 3'-gac aga ctt gc egg gtc ggc egg tac egg ! Scab.........Sfil............. ! NgoMl... t Ncol.... t ! FRl(DP47/V3-23)------------ ! 23 24 25 26 27 28 29 30

! EVQLLESG gaa|gtt|CAA|TTG|tta|gag|tct|ggt| 53 ! ctt|caa|gtt|aac|aat|ctc|aga|cca| ! | Mfel | ! ! -------------FR1-------------------------------- ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

! GGLVQPGGSLRLSCA ! |ccg|cca|gaajcaa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga| t ! Sites to be varied—> *** *** *** ! —FR1----------->|...CDR1................|—FR2----- ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

! ASGFTFSSYAMSWVR ! |cga|agg|cct|aag|tga|aag|agajagc|atg|cga|tac|aga$cc|caa|gcg| ! ] BspEI | | BsiWIj |BstXf. ! ! Sites to be varies—>*** ****** t ------FR2------------------->j...CDR2......... ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

! QAPGKGLEWVSAISG ! |gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|aga|cga|tag|agajcca| ! ...BstXI | t | *** *** ! .....CDR2............................................1—FR3— ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

! SGGSTYY ADSVKGRF |tctlegtlgeclagtiactltacltat|gct|gacltcc|gtt|aaa|ggt|cgc[ttc| 233 ! |aga|cca|ccg|tca|tga|atg|ata|cga|ctg]agg|caa|ttt|cca|gcg|aag| t ! ---FR3----------------------------- ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! TISRDNSKNTLYLQM |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| 278 . ! |tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac| ! | Xbal | t ! —FR3------------------------------>1 ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! NSLRAEDTAVYYCAK laaclagCITTAIAGg|pctlgag|gaclaCTIGCA|Gtc|tac|tatltgclgctlaaal 323 ! |ttg|tcg|aat|tcc|cga|ctc|ctg|tga|cgt|cag|atg|ata|acg|cga|ttt| ! IAflLI | | Pstl | ! .......CDR3.................|—FR4------------------ ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

! DYEGTGYAFDIWGQG |par|tatjpaalggtlactlggtltatlgctlttc|gaCIATAlTGg|ggt|caa|ggt] 368 ! |ctg|ata[ctt|cca]tga|cca|atafcga|aag|ctg|tat|acc|cca|gtt|cca| ! | Ndel | ! 1 ----------FR4-------->| ! 136 137 138 139 140 141142

! T Μ V T V S S |act|atG|GTC|ACC|gtc|tct]agt- 389 ! |tga|tac|cag|tgg|cag|aga|tca- ! | BstEII | i 143 144 145 146 147 148 149 150 151 152

! ASTKGPSVFP gcc tcc acc aaG GGC CCa teg GTC TTC ccc-3’ 419 ! egg agg tgg ttc ccg ggt age cag aag ggg-5' ! Bspl20I. Bbsl...(2/2) 1 Apal... (SFPRMET) 5'-ctg tet gaa cG GCC cag ccG-3’ (TOPFR1A) 5'-ctg tet gaa cG GCC cag ccG GCC atg gcc-gaa|gtt|CAA|TTG|tta|gag|tct|ggt|-lggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta-3' (BOTFR1B) 3'-caa|gtc|gga|cca|cca|aga|aatjgca|gaa|aga|acg|cga|- |cga|agg|ect|aag|tga|aag-5'! bottom strand (BOTFR2) 3'-acc|caa|gcg|- |gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|eaa|aga|-5' ! bottom strand (BOTFR3) 3’- a|cga|ctg|agg|caa|ttt|cca|gcg|aag|- |tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac|- |ttg|tcg|aat|tcc|ega|ctc|ctg|tga-5' (F06) 5’-gC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa|-|gac|tat|gaa|ggt|act|ggt|tat|gct|tte|gaC|ATA|TGg|ggt|c-3' (BOTFR4) 3'-cga|aag|ctg|tat|ace|cca|gtt|cca|-|tga|tac|cag|tgg|cag|aga|tca- egg agg tgg ttc ccg ggt age cag aag ggg-5'! bottom strand (BOTPRCPRIM) 3'-gg ttc ccg ggt age cag aag ggg-5' ! ! CDR1 diversity ! (ON-vgCl) 5'-lgctlTCCIGGA IttclactlttcItctK 1 >1TACI< 1 >|atg|< 1 >4 ! CDR1...................6859 ltgg|gtticgCICAa|gctlccTIGG-3' t !<1> stands for an equimolar mix of {ADEFGHIKLMNPQRSTVWY}; no C ! (this is not a sequence) t ! CDR2 diversity t (ON-vgC2) 5'-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>|- ! CDR2............ |tct|ggt|ggc|<l>|act|<l>|tat|gct|gac|tcc|gtt|aaa|gg-3' ! CDR2................................................

! <1> is an equimolar mixture of {ADEFGHIKLMNPQRSTVWY}; no C ! <2> is an equimolar mixture of {YRWVGS}; no ACDEFH1KLMNPQT

! <3> is an equimolar mixture of {PS}; no ACDEFGHIKLMNQRTVWY

Table 28: Stuffer used in VH

1 TCCGGAGCTT CAGATCTGTT TGCCTTTTTG TGGGGTGGTG CAGATCGCGT TACGGAGATC 61 GACCGACTGC TTGAGCAAAA GCCACGCTTA ACTGCTGATC AGGCATGGGA TGTTATTCGC 121 CAAACCAGTC GTCAGGATCT TAACCTGAGG CTTTTTTTAC CTACTCTGCA AGCAGCGACA 181 TCTGGTTTGA CACAGAGCGA TCCGCGTCGT CAGTTGGTAG A A AC ATT A AC ACGTTGGGAT 241 GGCATCAATT TGCTTAATGA TGATGGTAAA ACCTGGCAGC AGCCAGGCTC TGCCATCCTG 301 AACGTTTGGC TGACCAGTAT GTTGAAGCGT ACCGTAGTGG CTGCCGTACC TATGCCATTT 361 GATAAGTGGT ACAGCGCCAG TGGCTACGAA ACAACCCAGG ACGGCCCAAC TGGTTCGCTG 421 AATATAAGTG TTGGAGCAAA AATTTTGTAT GAGGCGGTGC AGGGAGACAA ATCACCAATC 481 CCACAGGCGG TTGATCTGTT TGCTGGGAAA CCACAGCAGG AGGTTGTGTT GGCTGCGCTG 541 GAAGATACCT GGGAGACTCT TTCCAAACGC TATGGCAATA ATGTGAGTAA CTGGAAAACA 601 CCTGCAATGG CCTTAACGTT CCGGGCAAAT AATTTCTTTG GTGTACCGCA GGCCGCAGCG 661 GAAGAAACGC GTCATCAGGC GGAGTATCAA AACCGTGGAA CAGAAAACGA TATGATTGTT 721 TTCTCACCAA CGACAAGCGA TCGTCCTGTG CTTGCCTGGG ATGTGGTCGC ACCCGGTCAG 781 AGTGGGTTTA TTGCTCCCGA TGGAACAGTT GATAAGCACT ATGAAGATCA GCTGAAAATG 841 TACGAAAATT TTGGCCGTAA GTCGCTCTGG TTAACGAAGC AGGATGTGGA GGCGCATAAG 901 GAGTCGTCTA GA

Table 29: DNA sequence of pCES5 ! pCES5 6680 bases = pCes4 with stuffers in CDRI-2 and CDR3 2000.12.13 ! ! Ngene = 6680 ! Useful REs (cut MAnoEl fewer than 3 times) 2000.06.05 !

Non-cutters !Acc651 Ggtacc Afel AGCgct AvrII Cctagg IBsaBl GATNNnnatc BsiWl Cgtacg BsmFI Nnnnnnnnnnnnnnngtccc IBsrGl Tgtaca BstAPI GCANNNNntgc BstBl TTcgaa !BstZ171 GTAtac Btrl CACgtg Eel 1361 GAGctc lEcoRV GATatc Fsel GGCCGGcc Kpnl GGTACc IMscl TGGcca Nrul TCGcga Nsil ATGCAt IPacl TTAATtaa Pmel GTTTaaac PmlI CACgtg IPpuMl RGgwccy PshAl GACNNnngtc Sacl GAGCTc ISacII CCGCgg Sbfl CCTGCAgg SexAI Accwggt ! Sgfl GCGATcgc SnaBl TACgta Spel Actagt ISphI GCATGc Sse8387l CCTGCAgg StulAGGcct ISwal ATTTaaat Xmal Cccggg | ! cutters ! Enzymes that cut more than 3 times. lAtwNI CAGNNNctg 5 IBsgl ctgcac 4 IBsrFI Rccggy 5 !Earl CTCTTCNnnn 4 IFaul nNNNNNNGCGGG 10 ! ! Enzymes that cut from 1 to 3 times.

I •EcoO 1091 RGgnccy 3 7 2636 4208 IBssSl Ctcgtg 1 12

Cacgag 1 1703 IBspHI Tcatga 3 43 148 1156 lAatll GACGTc 1 65 IBciVl GTATCCNNNNNN 2 140 1667 !Eco57I CTGAAG 1 301 cttcag 2 1349 'Aval Cycgrg 3 319 2347 6137 IBsiHKAI GWGCWc 3 401 2321 4245 IHgiAl GWGCWc 3 401 2321 4245 IBcgl gcannnnnntcg 1 461 'Seal AGTact 1 505 IPvuI CGATcg 3 616 3598 5926 IFspI TGCgca 2 763 5946 IBgll GCCNNNNnggc 3 864 2771 5952 IBpml CTGGAG 1 898 ctccag 1 4413 IBsal GGTCTCNnnnn 1 916 !Ahdl GACNNMnngtc 1 983 !Eaml 1051 GACNNNnngtc 1 983 IDrdl GACNNNNnngtc 3 1768 6197 6579 ISapI gaagage 1 1998 IPvull CAGetg 3 2054 3689 5896 IPflMl CCANNNNntgg 3 2233 3943 3991 IHindlfl Aagett 1 2235 !ApaLl Gtgeae 1 2321 IBspMI Nnnnnnnnngcaggt 1 2328 ACCTGCNNNMn 2 3460 IPstl CTGCAg 1 2335 !Accl GTmkac 2 2341 2611 IHincIl GTYrac 2 2341 3730 !Sall Gtegae 1 2341 !TliI Ctegag 1 2347 IXhoI Ctegag 1 2347 ! Bbsl gtctlc 2 2383 4219 IBIpI GCtnagc 1 2580 !EspI GCtnagc 1 2580 ISgrAI CReeggyg 1 2648 !Agel Accggt 2 2649 4302 lAscl GGcgcgcc 1 2689 IBssHH Gegege 1 2690 !SfiI GGCCNNNNnggcc 1 2770 •Nael GCCggc 2 2776 6349 INgoMIV Gccggc 2 2776 6349 !Btgl Ccrygg 3 2781 3553 5712 !Dsal Ccrygg 3 2781 3553 5712 INcol Ccatgg 1 2781 IStyl Ccwwgg 3 2781 4205 4472 IMfel Caattg I 2795 IBspEI Tccgga 1 2861 IBglll Agatct 1 2872 IBcil Tgatca 1 2956 !Bsu36I CCtnagg 3 3004 4143 4373 !XcmI CCANNNNNnnnntgg 1 3215 JMIul Acgcgt 1 3527 IHpal GTTaac 1 3730 IXbal Tclaga 1 3767 | 1ΑΠΙΙ Cttaag I 3811 !BsmI NGcattc 1 3821 GAATGCN 1 4695 IRsrll CGgwccg 1 3827 INhel Gctagc 1 4166 IBstEII Ggtnacc 1 4182 IBsmBI CGTCTCNnnnn 2 4188 6625

Nnnnnngagacg 1 6673 !Apal GGGCCc 1 4209 IBanll GRGCYc 3 4209 4492 6319 IBspI 201 Gggccc 1 4209 IPspOMl Gggccc 1 4209 IBseRI NNnnnnnnnnctcctc 1 4226 GAGGAGNNNNNNNNNN 1 4957 !EcoNI CCTNNnnnagg I 4278 IPfIFl GACNnngtc 1 4308 !Tthl 111 GACNnngtc 1 4308 !Kasl Ggcgcc 2 4327 5967 IBstXl CCANNNNNnlgg 1 4415 INotl GCggccgc 1 4507 lEagl Cggccg I 4508 IBamHI Ggatcc I 5169 IBspDI ATcgal I 5476 INdel CAtatg 1 5672 !EcoRl Gaattc 1 5806 IPsil TTAtaa 1 6118 IDralll CACNNNgtg 1 6243 IBsaAI YACgtr 1 6246 I____________________________________________________________________________ 1 gacgaaaggg cCTCGTGata cgcctatttt tataggttaa tgtcalgata ataatggttt ! BssSl.(l/2) 61 cttaGACGTC aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt ! Aatll. 121 tctaaataca ttcaaatatG TATCCgctca tgagacaata accctgataa atgcttcaat ! BciVI..(l of 2) 181 aatattgaaa aaggaagagt 1 Base # 201 to 1061 = ApR gene from pUCl 19 with some RE sites removed ! ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

! fMSIQHFRVALIPFFA 201 atg agt att caa cat ttc cgt gtc gee ett att ccc ttt ttt geg

I 1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

! AFCLPVFAHPETLVK 246 gca ttt tgc ett cct gtt ttt get cac cca gaa aeg ctg gtg aaa ! ! 31 32 33 34 35 36 37 38 39 40 4! 42 43 44 45

! VKDAEDQLGARVGYI 291 gta aaa gat get gaa gat cag ttg ggt gee ega gtg ggt tac ate ! ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

! ELDLNSGKILESFRP 336 gaa ctg gat etc aac age ggt aag ate ett gag agt ttt ege ccc ! ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

! EERFPMMSTFKVLLC 381 gaa gaa cgt ttt cca atg atg age act ttt aaa gtt ctg eta tgt

I ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 ! gavlsridagqeqlg 426 ggc gcg gta tta tee cgt att gac gee ggg caa gaG CAa etc ggT ! Bcgl............ ! ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! RRIHYSQNDLVEYSP 471 CGc ege ata cac tat tet cag aat gac ttg gtt gAG TAC Tea cca !..Bcgl...... Seal....

I ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! VTEKHLTDGMTVREL 516 gtc aca gaa aag cat ett aeg gat ggc atg aca gta aga gaa tta ! ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

! CSAA1TMSDNTAANL 561 tgc agt get gee ata ace atg agt gat aac act gcg gee aac tta ! I 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150

! LLTTIGGPKELTAFL 606 ett ctg aca aCG ATC Gga gga ccg aag gag eta ace get ttt ttg ! Pvul... (1/2) ! ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

! HNMGDHVTRLDRWEP 651 cac aac atg ggg gat cat gta act ege ett gat cgt tgg gaa ccg j ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

! ELNEA1PNDERDTTM 696 gag ctg aat gaa gee ata cca aac gac gag cgt gac ace aeg atg j ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

! PVAMATTLRKLLTGE 741 cct gta GCA ATG gca aca aeg tTG CGC Aaa eta tta act ggc gaa ! BsrDI..(1/2) Fspl.... (1/2) ! ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

! LLTLASRQQLIDWME 786 eta ett act eta get tee egg caa caa tta ata gac tgg atg gag ! ! 211 212213 214 215 216 217218 219 220 221 222 223 224 225

! ADKVAGPLLRSALPA 831 geg gat aaa gtt gca gga cca ett ctg ege teg gee ett ccg get ! ! 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

1 GWFIADKSGAGERGS 876 ggc tgg ttt att get gat aaa tCT GGA Gee ggt gag cgt gGG TCT ! Bpml....(1/2) Bsal.... t ! 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

! RGIIAALGPDGKPSR 921 Cgc ggt atC ATT GCa gca ctg ggg cca gat ggt aag ccc tee cgt ! Bsal...... BsrDI...(2/2) t ! 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270

! IVV1YTTGSQATMDE 966 ate gta gtt ate tac aeG ACg ggg aGT Cag gca act atg gat gaa ! Ahdl........... ! ! 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285

! RNRQ1AE1GASLIKH 1011 ega aat aga cag ate get gag ata ggt gee tea ctg att aag cat ! ! 286 287 ! W . 1056 tgg taa 1062 ctgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatett cttgagatcc tttttttctg egegtaatet 1261 getgettgea aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct teageagage gcagatacca aatactgtcc 1381 ttctagtgta geegtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 5 561 cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggaeagGTAT CCggtaagcg ! BciVI.. (2 of 2) 1681 gcagggtcgg aacaggagag cgCACGAGgg agcttccagg gggaaacgcc tggtatcttt ! BssSI.(2/2) 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt ttlacggttc ctggcctttt 1861 gctggccttt tgclcACATG Ttctttcctg cgttatcccc tgattctgtg gataaccgta ! Pcil... 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcgG AA G AGCgcccaa tacgcaaacc gcctctcccc gcgcgttggc ! Sap!.... 2041 cgattcatta atgCAGCTGg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca ! PvuII.(l/3) 2101 acgcaatTAA TGTgagttag ctcactcatt aggcacccca ggcTTTACAc tttatgcttc ! ,.-35.. Plac ..-10.

2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacaCAGGA AACAGCTATG ! Μ13Rev_seq_primer 2221 ACcatgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac ! PflMI....... ! Hind3. ! signal::linker::CLight | ! 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15

! fMKKLLFAIPLVVPFY 2269 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat ] 1 Linker..............................End of FR4 ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 ! SHSA QVQLQVDLEIK 2314 tct cac aGT GCA Cag gtc caa CTG CAG GTC GAC CTC GAG ate aaa ! ApaLI...... Pstl... Xhol... ! BspMl... i Sail... 1 Accl...(1/2) ! Hindi.(1/2) ! ! Vlight domains could be cloned in as ApaLI-Xhol fragments. 1 VL-CL(kappa) segments can be cloned in as ApaLI-AscI fragments. <-------- ! ! Ckappa---------------------------------------------------- 1 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

! RGTVAAPSVF1FPPS 2359 cgt gga act gtg get gca cca tet GTC TTC ate tie ccg cca tet ! Bbsl...(1/2) ! ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

! DEQLKSGTASVVCLL 2404 gat gag cag ttg aaa tet gga act gee tet gtt gtg tgc ctg ctg ; ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

! NNFYPREAKVQWKVD 2449 aat aac ttc tat ccc aga gag gee aaa gta cag tgg aag gtg gat ! ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

! NALQSGNSQESVTEQ 2494 aac gee etc caa teg ggt aac tee cag gag agt gtc aca gag cag ! ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! DSK.DSTYSLSSTLTL 2539 gac age aag gac age ace tac age etc age age ace ctg aeG CTG ! Espl... ! ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! SKADYEKHKVYACEV 2584 AGC aaa gca gac tac gag aaa cac aaa GTC TAC gee tgc gaa gtc ! ...Espl.... Accl...(2/2) ! ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

! THQGLSSPVTK.SFNR 2629 acc cat cag ggc ctg agt tcA CCG GTg aca aag age ttc aac agg ! Agel....(l/2) ! ! 136 137 138 139 140 1 G E C . . 2674 gga gag tgt taa taa GG CGCGCCaatt ! AscI..... ! BssHIl. ! 2701 ctatttcaag gagacagtca ta ! ! PelB::3-23(stuffed)::CHl ::111 fusion gene ! ! 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15

! MK.YLLPTAAAGLLLL 2723 atg aaa tac eta ttg cct aeg gca gcc get gga ttg tta tta etc

I !____________________________________________ 1 ! 16 17 18 19 20 21 22 ! A A Q P A M A 2768 geG GCC cag ccG GCC atg gcc ! Sfil............. ! NgoMiV..(l/2) ! Ncol.... ! ! FRI(DP47/V3-23)--------------- ! 23 24 25 26 27 28 29 30

! EVQLLESG 2789 gaa|gtt|CAA|TTG|tta|gag|tct|ggt| ! | Mfel | ! ! FR1-------------------------------------------- ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 ! GGLVQPGGSLRLSCA 2813 |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| | ! —FR1----- ! 46 47 48

!. A S G 2858 |gct|TCC|GGA| ! | BspEI|

I ! Sniffer for CDRI, FR2, and CDR2---------------------------------> ! There are no stop codons in this stuffer. 2867 gcttcAGATC Tgtttgcctt ! Bglll.. 2887 tttgtggggt ggtgcagatc gcgttacgga gatcgaccga ctgcttgagc aaaagccacg 2947 cttaactgcT GATCAggcat gggatgttat tcgccaaacc agtcgtcagg atcttaacct ! Bell... 3007 gaggcttttt ttacctactc tgcaagcagc gacatctggt ttgacacaga gcgatccgcg 3067 tcgtcagttg gtagaaacat taacacgttg ggatggcatc aatttgctta atgatgatgg 3127 taaaacctgg cagcagccag gctctgccat cctgaacgtt tggclgacca gtatgttgaa 3187 gcgtaccgta gtggctgccg tacctatgCC Atttgataag TGGtacagcg ccagtggcta ! Xcml............. 3247 cgaaacaacc caggacggcc caactggttc getgaatata agtgttggag caaaaatttt 3307 gtatgaggcg gtgcagggag acaaatcace aatcccacag gcggttgatc tgtttgctgg 3367 gaaaccacag caggaggttg tgttggctgc gctggaagat acctgggaga ctctttccaa 3427 aegetatgge aataatgtga gtaactggaa aacacctgca atggccttaa cgttccgggc 3487 aaataatttc tttggtgtac cgcaggccgc ageggaagaa ACGCGTcatc aggeggagta ! MIuI.. 3547 tcaaaaccgt ggaacagaaa aegatatgat tgttttctca ccaacgacaa gcgatcgtcc 3607 tgtgcttgcc tgggatgtgg tcgcacccgg tcagagtggg tttattgctc ccgatggaac 3667 agttgataag cactatgaag atcagctgaa aatgtacgaa aattttggcc gtaagteget ! Pvull. 3 727 ctgGTTAACg aagcaggatg tggaggegea taaggagteg ! Hpal.. ! HincII(2/2) ! ! --------FR3-------------------------------------------------- ! 4 5 6 7 8 9 10 II 12 13 14 15 16 I 93 94 95 96 97 98 99 100 101 102 103 104 105

! SRDNSKNTLYLQM 3767 |TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| ! I Xbal |

I ! —FR3----------------------------------------------------->| ! 17 18 19 20 ! 106 107 108 109 ! NSLsIsirsg 3806 |aac|agC|TTA|AG t ctg age att CGG TCC G ! | Afl 111 RsrII.. ! ! q h s p t .

3834 gg caa cat tet cca aac tga ccagacga cacaaacggc 3872 ttacgctaaa tcccgcgcat gggatggtaa agaggtggcg tctttgctgg cctggactca 3932 tcagatgaag gccaaaaatt ggcaggagtg gacacagcag gcagcgaaac aagcactgac 3992 catcaactgg tactatgctg atgtaaacgg caatattggt tatgttcata ctggtgctta 4052 tccagatcgt caatcaggcc atgatccgcg attacccgtt cctggtacgg gaaaatggga 4112 ctggaaaggg etattgeett ttgaaatgaa ccctaaggtg tataaccccc ag 4164 aa GCT AGC ctgcggcttc ! NheL ! 4182 G|GTC|ACC| gtctcaagc ! | BstEIl | ! ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150

! ASTKGPSVFPLAPSS 4198 gee tee ace aag ggc cca teg gtc ttc ccc ctg gca ccc tee tee ! ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

! KSTSGGTAALGCLVK 4243 aag age 1. t ggg ggc aca geg gee ctg ggc tgc ctg gtc aag j ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

! DYFPEPVTVSWNSGA 4288 gac tac ttc ccc gaa ccg gtg aeg gtg teg tgg aac tea ggc gee ! ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

! LTSGVHTFPAVLQSS 4333 ctg acc age ggc gtc cac acc ttc ccg get gtc eta cag tee tea ! ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

! GLYSLSSVVTVPSSS 4378 gga etc tac tee etc age age gta gtg acc gtg ccc tee age age t ! 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225

! LGTQTY1CNVNHKPS 4423 ttg ggc acc cag acc tac ate tgc aac gtg aat cac aag ccc age j ! 226 227 228 229 230 231 232 233 234 235 236 237 238

! NTKVDKKVEPKSC 4468 aac acc aag gig gac aaG AAA GTT GAG CCC AAA TCT TGT ! ON-TQHCforw...................... ! Poly His linker ! 139 140 141 142 143 144 145 146 147 148 149 150

! AAAHHHHHHGAA 4507 GCG GCC GCa cat cat cat cac cat cac ggg gee gca ! Noll...... ! Eagl.... ! ! 151 152 153 154 155 156 157 158 159 !60 161 162 163 164 165 ! EQKLISEEDLNGAA.

4543 gaa caa aaa etc ate tea gaa gag gat ctg aat ggg gee gca tag I ! Mature 111------------------------------------------------>— ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

t TVESCLAKPHTENSF 4588 act gtt gaa agt tgt tta gca aaa cct cat aca gaa aat tea ttt t ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

! TNVWKDDKTLDRY AN 4633 act aac gtc tgg aaa gac gac aaa act tta gat cgt tac get aac ! ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

! YEGCLWNATGVVVCT 4678 lat gag ggc tgt ctg tgG AAT GCt aca ggc gtt gtg gtt tgt act ! BsmI.... ! ! 211 212 213 214215 216217218 219220221222223 224225

! GDETQCYGTWVP1GL 4723 ggt gac gaa act cag tgt tac ggt aca tgg gtt cct att ggg clt t ! 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

! AIPENEGGGSEGGGS 4768 get ate cct gaa aat gag ggt ggt ggc tet gag ggt ggc ggt tet j ! 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

! EGGGSEGGGTKPPEY 4813 gag ggt ggc ggt tet gag ggt ggc ggt act aaa cct cct gag tac ! 1 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270

! GDTP1PGYTYINPLD 4858 ggt gat aca cct att ccg ggc tat act tat ate aac cct etc gac 1 ! 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285

! GTYPPGTEQNPANPN 4903 ggc act tat ccg cct ggt act gag caa aac ccc get aat eel aat ] ! 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

! PSLEESQPLNTFMFQ 4948 cct tet ett GAG GAG tet cag cct ett aat act ttc atg ttt cag ! BseRI..(2/2) t ! 301 302 303 304 305 306 307 308 309 310 311 312313 314315

! NNRFRNRQGALTVYT 4993 aat aat agg ttc ega aat agg cag ggt gca tta act gtt tat aeg ! ! 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330

! GTVTQGTDPVK.TYYQ 5038 ggc act gtt act caa ggc act gac ccc gtt aaa act tat tac cag ! ! 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 I YTPVSSKAMYDAYWN 5083 tac act cct gta tea tea aaa gee atg tat gac get tac tgg aac !

i 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 ! GKFRDCAFHSGFNED 5128 ggt aaa ttc aga gac tgc get ttc cat tet ggc ttt aat gaG GAT ! BamHI.. ! ! 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375

! PFVCEYQGQSSDLPQ 5173 CCa ttc gtt tgt gaa tat caa ggc caa teg tet gAC CTG Cct caa ! BamHI... BspMI...(2/2) ! ! 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

! PPVNAGGGSGGGSGG 5218 cct cct gtc aat get ggc ggc ggc tet ggt ggt ggt tet ggt ggc ! ! 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

! GSEGGGSEGGGSEGG 5263 ggc tet gag ggt ggc ggc tet gag ggt ggc ggt tet gag ggt ggc

I ! 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

! GSEGGGSGGGSGSGD 5308 ggc tet gag ggt ggc ggt tee ggt ggc ggc tee ggt tee ggt gat j ! 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 ! fdyekmanankgamt 5353 ttt gat tat gaa aaa atg gca aac get aat aag ggg get atg acc j ! 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450

! ENADENALQSDAKGK 5398 gaa aat gee gat gaa aac geg eta cag tet gac get aaa ggc aaa t ! 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 ! LDSVATDYGAAIDGF 5443 ctt gat tct gtc get act gat tac ggt get get ATC GAT ggt ttc ! BspDI.. ! ! 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

! 1GDVSGLANGNGATG 5488 att ggt gac gtt tee ggc ctt get aat ggt aat ggt get act ggt

I ! 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

! DFAGSNSQMAQVGDG 5533 gat ttt get ggc tct aat tee caa atg get caa gtc ggt gac ggt | ! 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

! DNSPLMNNFRQYLPS 5578 gat aat tea cct tta atg aat aat ttc cgt caa tat tta cct tct ! ! 511 512513 514515516517518519 520 521 522 523 524 525

! LPQSVECRPYVFGAG 5623 ttg cct cag teg gtt gaa tgt ege cct tat gtc ttt ggc get ggt ! ! 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

! KPYEFSIDCDK1NLF 5668 aaa cCA TAT Gaa ttt tct att gat tgt gac aaa ata aac tta ttc ! Ndel.... j ! 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555

! RGVFAFLLYVATFMY 5713 cgt ggt gtc ttt geg ttt ctt tta tat gtt gee acc ttt atg tat j ! 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 ! VFSTFANiLRNK.ES. 5758 gta ttt teg aeg ttt get aac ata ctg cgt aat aag gag tct taa ! ! 571 ! 5803 taa GAATTC ! EcoRI.

5812 actggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 5871 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcacCGATC ! Pvul.. 5931 Gcccttccca acagtTGCGC Agcctgaatg gcgaatGGCG CCtgatgcgg tattttctcc ! ...Pvul... (3/3) Fspl... (2/2) KasI...(2/2) 5991 ttacgcatct gtgcggtatt tcacaccgca tataaattgt aaacgttaat atutgttaa 6051 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 6111 aaatcccTTA TAAatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga ! Psil... 6171 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 6231 agggcgatgg ccCACtacGT Gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc ! Dralll.... 6291 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaaGC ! NgoMiV.. 6351 CGGCgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg ! ..NgoMiV.(2/2) 6411 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 6471 agggcgcgla ctatggttgc tttgacgggt gcaglctcag tacaatctgc tctgatgccg 6531 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 6591 tgctcccggc atccgcttac agacaagctg Igaccgtctc cgggagctgc atgtgtcaga 6651 ggttttcacc gtcatcaccg aaacgcgcga

Table 30: Oligonucleotides used to clone CDR1/2 diversity All sequences are 5' to 3’. 1) ON CDl Bsp, 30 bases

AccTcAcTggcTTccggA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 TTcAcTTT cTcT 19 20 21 22 23 24 25 26 27 28 29 30 2) ON_Brl2,42 bases

AgAAAcccAcTccAAAcc 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 TTTAccAggAgcTTggcg 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 A A c c c A 37 38 39 40 41 42 3) ON_CD2Xba, 51 bases ggAAggcAgTgATcT AgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 g A T A g T gAAgcgAccTTT 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 AAcggAgTcAgcATA 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 4) ON BotXba, 23 bases ggAAggcAgTgATcTAgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 g A T A g 19 20 21 22 23

Table 31: Bridge/Extender Oligonucleotides ON LamlaB7(rc) .........................GTGCTGACTCAGCCACCCTC. 20 ON~Lam2aB7 (rc) ........................GCCCTGACTCAGCCTGCCTC. 20 ON Lam31B7(rc) .......................GAGCTGACTCAGG.ACCCTGC 20 ON Lam3rB7 (rc) ........................GAGCTGACTCAGCCACCCTC. 20 ON~LamHflcBrg{rc) CCTCGACAGCGAAGTGCACAGAGCGTCTTGACTCAGCC....... 38 ON_LamHf 1 cExt CCTCGACAGCGAAGTGCACAGAGCGTCTTG............... 30 ON_LamHf2b2Brg (rc) CCTCGACAGCGAAGTGCACAGAGCGCTTTGACTCAGCC....... 38 ON_LamHf2b2Ext CCTCGACAGCGAAGTGCACAGAGCGCTTTG............... 30

ON_LamHf2dBrg (rc) CCTCGACAGCTAAGTGCACAGAGCGCTTTGACTCAGCC....... 3B ON_LamHf2dExt CCTCGACAGCGAAGTGCACAGAGCGCTTTG............... 30 ON_LamHf31Brg (rc) CCTCGACAGCGAAGTGCACAGAGCGAATTGACTCAGCC....... 38 ON_LamHf 31Ext CCTCGACAGCGAAGTGCACAGAGCGAATTG............... 30 ON_LamHf3rBrg (rc) CCTCGACAGCGAAGTGCACAGTACGAATTGACTCAGCC....... 38 ON_LamHf 3rExt CCTCGACAGCGAAGTGCACAGTACGAATTG............... 30 ON_lamPlePCR CCTCGACAGCGAAGTGCACAG........................ 21

Consensus

Table 32: Oligonucleotides used to make SSDNA locally double-stranded

Adapters (8) H43HF3.1?02#1 5'-cc gtg tat tac tgt gcg aga g-3' H43.77.97.l-03#2 5'-ct gtg tat tac tgt gcg aga g-3' H43.77.97.323#22 5 ’-CC gtl tat tac tgt gcg a|a g-3’ H43.77.97.330#23 5'-cl gtg tat tac tgt gcg a@a g-3' H4 3.77.97.439#44 5'-cj| gtg tat tac tgt gcg aga S“3' H4 3.77.97.551#48 5 *-cc §tg tat tac tgt gcg aga j§-3'

Table 33: Bridge/extender pairs

Bridges (2) H4 3.XABrl 5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg ctgaggacaCTGCAGtctactattgtgcgaga-3' H4 3.XABr2 5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg ctgaggacaCTGCAGtctactattgtgcgaaa-3'

Extender H4 3.XAExt 5' ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTcATcTgcAAgTAgAgAgTATTcTTAg AgTTgTcTcTAgATcAcTAcAcc-3'

Table 34: PCR primers Primers H43.XAPCR2 gactgggTgTAgTgATcTAg

Hucmnest cttttctttgttgccgttggggtg

Table 35: PCR program for amplification of heavy chain CDR3 DNA 95 degrees C 5 minutes 95 degrees C 20 seconds 60 degrees C 30 seconds repeat 20x 72 degrees C 1 minute 72 degrees C 7 minutes 4 degrees C hold

Reagents (100 ul reaction):

Template 5ul ligation mix lOx PCR buffer lx

Taq 5U dNTPs 200 uM each

MgCl2 2mM

H43.XAPCR2-biotin 400 nM

Hucmnest 200 nM ! Table 36; Annotated sequence of CJR DY3F7(CJR-A05) 10251 bases j ! Non-cutters i '.Bell Tgatca BsiWI Cgtacg BssSI Cacgag !BstZ17I GTAtac Btrl CACgtg EcoRV GATatc ! Fsel GGCCGGcc Hpal GTTaac Mlul Acgcgt !Pmel GTTTaaac Pmll CACgtg PpuMI RGgwccy !RsrII CGgwccg SapI GCTCTTC SexAI Accwggt !SgfI GCGATcgc SgrAI CRccggyg SphI GCATGc !StuI AGGcct Xraal Cccggg

I ! cutters i ! Enzymes that cut from 1 to 4 times and other features

I !End of genes II and X 829 !Start gene V 843 !BsrGI Tgtaca 1 1021 !BspMI Nnnnnnnnngcaggt 3 1104 5997 9183 ACCTGCNNNNn 1 2281 !End of gene V 1106 ! Start gene VII- > 1108 !BsaBI GATNNnnatc 2 1149 3967 !Start gene IX 1208 !End gene VII 1211 !SnaBI TACgta 2 1268 7133 !BspHI Tcatga 3 1299 6085 7093 !Start gene VIII 1301 !End gene IX 1304 !End gene VIII 1522 IStart gene III 1578 !EagI Cggccg 2 1630 8905 !Xbal Tctaga 2 1643 8436 !KasI Ggcgcc 4 1650 8724 9039 9120 !BsmI GAATGCN 2 1769 9065 · IBseRI GAGGAGNNNNNNNNNN 2 2031 8516 NNnnnnnnnnctcctc 2 7603 8623 !AlwNI CAGNNNctg 3 2210 8072 8182 !BspDl ATcgat 2 2520 9883 INdel CAtatg 3 2716 3796 9847 !End gene III 2846 IStart gene VI 2848 !Afel AGCgct 1 3032 !End gene VI 3187 IStart gene I 3189 I Earl CTCTTCNnnn 2 4 067 9274

Nnnnngaagag 2 6126 8953 IPacI TTAATtaa 1 4125 IStart gene IV 4213 I End gene I 4 235 IBsmFI Nnnnnnnnnnnnnnngtccc 2 5068 9515 IMscI TGGcca 3 5073 7597 9160 IPsil TTAtaa 2 5349 5837 I End gene IV 54 93 IStart ori 5494 INgoMIV Gccggc 3 5606 8213 9315 IBanll GRGCYc 4 5636 8080 8606 8889 IDralll CACNNNgtg 1 5709 IDrdI GACNNNNnngtc 1 5752 lAval Cycgrg 2 5818 7240 IPvuII CAGctg 1 5953 !BsmBI CGTCTCNnnnn 3 5964 8585 9271 !End ori region 5993 !BamHI Ggatcc 1 5994 !Hindlll Aagctt 3 6000 7147 7384 !BciVI GTATCCNNNNNN 1 607 7 , ' · !Start bla 6138 !Eco57I CTGAAG 2 6238 7716 !SpeI Actagt 1 6257 !BcgI gcannnnnntcg 1 6398 !Seal AGTact . 1 6442 !Pvul CGATcg 1 6553 !FspI TGCgca 1 6700 !Bgll GCCNNNNnggc 3 6801 8208 8976 !Bsal GGTCTCNnnnn 1 6853 . !Ahdl GACNNNnngtc 1 6920 !Eamll05I GACNNNnngtc 1 6920 !End bla 6998 !AccI GTmkac 2 7153 8048 .'Hindi GTYrac 1 7153 !Sail Gtcgac 1 7153 !XhoI Ctcgag 1 7240 !Start PlacZ region 7246 !End PlacZ region 7381 IPflMI CCANNNNntgg 1 7382 !RBS1 7405

Istart M13-iii signal seq for LC 7418 lApaLI Gtgcac 1 7470 lend M13-iii signal seq 7471 !Start light chain kappa L20:JK1 7472 IPflFI GACNnngtc 3 7489 8705 9099 !SbfI CCTGCAgg 1 7542 !PstI CTGCAg 1 7543 !Kpnl GGTACc 1 7581 !Xcml CCANNNNNnnnntgg 2 7585 9215 !Nsil ATGCAt 2 7626 9503 !BsgI ctgcac 1 7809 !Bbsl gtette 2 7820 8616 !BlpI GCtnagc 1 8017 !EspI GCtnagc 1 8017 !Eco0109I RGgnccy 2 8073 8605 !Ecll36I GAGctc 1 8080 !SacI GAGCTc 1 8080 !End light chain 8122 !Ascl GGcgcgcc 1 8126 !BssHII Gegege 1 8127 !RBS2 8147 !Sfil GGCCNNNNnggcc 1 8207 !Ncol Ccatgg 1 8218 IStart 3-23, FR1 8226 IMfel Caattg 1 8232 IBspEI Teegga 1 8298 IStart CDR1 8316 IStatt FR2 8331 IBstXI CCANNNNNntgg 2 8339 8812 lEcoNI CCTNNnnnagg 2 8346 8675 IStart FR3 8373 IXbal Tetaga 2 8436 1643 lAflll Cttaag 1 8480 IStart CDR3 8520 lAatll GACGTc 1 8556 ! Start FR4 8562 !PshAI GACNNnngtc 2 8573 9231 !BstEII Ggtnacc 1 8579 !Start CHI 8595 !Apal GGGCCc 1 8606 !Bspl20I Gggccc 1 8606 !PspOMI Gggccc 1 8606 !AgeI Accggt 1 8699 !Bsu36I CCtnagg 2 8770 9509 !End of CHI 8903 !Notl GCggccgc 1 8904 'Start His6 tag 8913 'Start cMyc tag 8931 'Amber codon 8982 !NheI Gctagc 1 8985 'Start M13 III Domain 3 8997 !Nrul TCGcga 1 9106 !BstBI TTcgaa 1 9197 !EcoRI Gaattc 1 9200 !Xcml CCANNNNNnnnntgg 1 9215 !BstAPI GCANNNNntgc 1 9337 ! SacII CCGCgg 1 9365 ! End Illstump anchor 9455 !AvrII Cctagg 1 9462 ! trp terminator 9470 !Swal ATTTaaat 1 9784 !Start gene II 9850 !Bglll Agatct 1 9936 I---------------------------------------------------------------------- 1 aat get act act att agt aga att gat gcc acc ttt tea get ege gee ! gene ii continued 49 cca aat gaa aat ata get aaa cag gtt att gac cat ttg ega aat gta 97 tet aat ggt caa act aaa tet act cgt teg cag aat tgg gaa tea act 145 gtt aTa tgg aat gaa act tee aga cac cgt act tta gtt gca tat tta 193 aaa cat gtt gag eta cag caT TaT att cag caa tta age tet aag cca 241 tee gca aaa atg acc tet tat caa aag gag caa tta aag gta etc tet 289 aat cct gac ctg ttg gag ttt get tee ggt ctg gtt egc ttt gaa get 337 ega att aaa aeg ega tat ttg aag tet ttc ggg ett cct ett aat ett 385 ttt gat gca ate ege ttt get tet gac tat aat agt cag ggt aaa gac 433 ctg att ttt gat tta tgg tea ttc teg ttt tet gaa ctg ttt aaa gca 481 ttt gag ggg gat tea ATG aat att tat gac gat tee gca gta ttg gac ! Start gene x, ii continues 529 get ate cag tet aaa cat ttt act att acc ccc tet ggc aaa act tet 577 ttt gca aaa gcc tet ege tat ttt ggt ttt tat cgt cgt ctg gta aac 625 gag ggt tat gat agt gtt get ett act atg cct cgt aat tee ttt tgg 673 cgt tat gta tet gca tta gtt gaa tgt ggt att cct aaa tet caa ctg 721 atg aat ett tet acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att 769 aac gta gat ttt tet tee caa cgt cct gac tgg tat aat gag cca gtt

817 ett aaa ate gca TAA

! End X &amp; II 832 ggtaattca ca ! ! Ml ' E5 Q10 T15 843 ATG att aaa gtt gaa att aaa cca tet caa gcc caa ttt act act cgt

! Start gene V

I ! S17 S20 P25 E30 891 tet ggt gtt tet cgt cag ggc aag cct tat tea ctg aat gag cag ett ] ! V35 E40 V45 939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ett gtc aag att act

I ! D50 A55 L60 987 ctt gat gaa ggt cag cca gcc tat gcg cct ggt cTG TAC Acc gtt cat ! BsrGI. . . ! L65 V70 S75 R80 1035 ctg tcc tct ttc aaa gtt ggt cag ttc ggt tcc ctt atg att gac cgt

I

! P85 K87 end of V

1083 ctg cgc etc gtt ccg get aag TAA C

I

1108 ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg ! Start gene VII

I 1150 ata caa ate tcc gtt gta ctt tgt ttc gcg ctt ggt ata ate

I ! VII and IX overlap. ! ..... S2 V3 L4 V5 S10 1192 get ggg ggt caa agA TGA gt gtt tta gtg tat tct ttT gcc tct ttc gtt

! End VII

! I start IX ! L13 W15 G20 T25 E29 1242 tta ggt tgg tgc ctt cgt agt ggc att aeg tat ttt acc cgt tta atg gaa

I 1293 act tcc tc i ! .... stop of IX, IX and VIII overlap by four bases 1301 ATG aaa aag tct tta gtc etc aaa gcc tct gta gcc gtt get acc etc ! Start signal sequence of viii. i 1349 gtt ccg atg ctg tct ttc get get gag ggt gac gat ccc gca aaa gcg ! mature VIII---> 1397 gcc ttt aac tcc ctg caa gcc tea gcg acc gaa tat ate ggt tat gcg 1445 tgg gcg atg gtt gtt gtc att 1466 gtc ggc gca act ate ggt ate aag ctg ttt aag ! ! bases 1499-1539 are probable promoter for iii 1499 aaa ttc acc teg aaa gca ! 1515 ! ........... -35 . . i 1517 age tga taaaccgat acaattaaag gctccttttg ! ..... -10 ... ' i 1552 gagccttttt ttt GGAGAt ttt ! S.D. uppercase, there may be 9 Ts 1 i <------ill signal sequence----------------------------->

! MKKLLFAIPLVVPF 1574 caac GTG aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc ! 1620 i

! YSGAAES HLDGA 1620 tat tct ggc gCG GCC Gaa tea caT CTA GAc ggc gcc ! EagI.... Xbal.... 1 ! Domain 1 ------------------------------------------------------------

! AETVESCLA 1656 get gaa act gtt gaa agt tgt tta gca t

! KSHTEISFTNVWKDDKT 1683 aaA Tcc cat aca gaa aat tea ttt aCT AAC GTC TGG AAA GAC GAC AAA ACt

I

! LDRYANY EGSLWNATG V 1734 tta gat cgt tac get aac tat gag ggC tgt ctg tgG AAT GCt aca ggc gtt ! BsmI....

I

! VVCTGDETQCYGTWVPI 1785 gta gtt tgt act ggt GAC GAA ACT CAG TGT TAC GGT ACA TGG GTT cct att i

! G L A I P E N 1836 ggg ctt get ate cct gaa aat ! ! LI linker ------------------------------------

! EGGGSEGGGS 1857 gag ggt ggt.ggc tet gag ggt ggc ggt tet

I

! EGGGSEGGGT 1887 gag ggt ggc ggt tet gag ggt ggc ggt act

I ! Domain 2 ------------------------------------ 1917 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat ate aac 1968 cct etc gac ggc act tat ccg cct ggt act gag caa aac ccc get aat cct 2019 aat cct tet ctt GAG GAG tet cag cct ctt aat act ttc atg ttt cag aat ! BseRI.. 2070 aat agg ttc ega aat agg cag ggg gca tta act gtt tat aeg ggc act 2118 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct

2166 gta tea tea aaa gee atg tat gac get tac tgg aac ggt aaa ttC AGA

! AlwNI

2214 GAC TGc get ttc cat tet ggc ttt aat gaG gat TTa ttT gtt tgt gaa ! AlwNI 2262 tat caa ggc caa teg tet gac ctg cct caa cct cct gtc aat get i 2307 ggc ggc ggc tet ! start L2 ------------------------------------------------------------- 2319 ggt ggt ggt tet 2331 ggt ggc ggc tet 2343 gag ggt ggt ggc tet gag gga ggc ggt tee 2373 ggt ggt ggc tet ggt ! end L2

I ! Many published sequences of M13-derived phage have a longer linker ! than shown here by repeats of the EGGGS motif two more times.

I ! Domain 3 --------------------------------------------------------------

! SGDF DYEKMANANKGA 2388 tee ggt gat ttt gat tat gaa aag atg gca aac get aat aag ggg get |

! MTENADENALQSDAKG 2436 atg acc gaa aat gee gat gaa aac geg eta cag tet gac get aaa ggc i

! KLDSVATDYGAAMDGF 2484 aaa ctt gat tet gtc get act gat tac ggt get get ate gat ggt ttc i

! IGDV SGLANGNGATGD 2532 att ggt gac gtt tee ggc ctt get aat ggt aat ggt get act ggt gat

! FAGS NSQMAQVGDGDN 2580 ttt get ggc tet aat tee caa atg get caa gtc ggt gac ggt gat aat i

! SPLMNNFRQYLPSLPQ 2628 tea cct tta atg aat aat ttc cgt caa tat tta cct tee etc cct caa !

! SVEC RPFVFGAGKPYE 2676 teg gtt gaa tgt ege cct ttt gtc ttt Ggc get ggt aaa cca tat gaa

I

! FSIDCDKINLFR 2724 ttt tct att gat tgt gac aaa ata aac tta ttc cgt ! End Domain 3 1 I G V F A F LLYV A T F Μ Y V F140 2760 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt ! start transmembrane segment i

! S T F A N I L 2808 tct acg ttt get aac ata ctg

I

! R N K E S 2829 cgt aat aag gag tct TAA ! stop of iii ! Intracellular anchor.

I ! Ml P2 V L L5 G I P L L10 L R F L G15

2847 tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc etc ggt ! Start VI i 2894 ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag 2942 ggc ttc ggt aag ata get att get att tea ttg ttt ctt get ctt att 2990 att ggg ctt aac tea att ctt gtg ggt tat etc tct gat att age get 3038 caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att etc ccg tct 3086 aat gcg ctt ccc tgt ttt tat gtt att etc tct gta aag get get att 3134 ttc att ttt gac gtt aaa caa aaa ate gtt tct tat ttg gat tgg gat ! ! Ml A2 V3 F5 L10 G13

3182 aaa TAA t ATG get gtt tat ttt gta act ggc aaa tta ggc tct gga ! end VI Start gene I

I

! KTLVS VGKI Q DKIVA 3228 aag acg etc gtt age gtt ggt aag att cag gat aaa att gta get |

! GCKIATNLDLRLQNL 3273 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac etc 1

! PQVGR FAKT PRVLRI 3318 ccg caa gtc ggg agg ttc get aaa acg cct ege gtt ctt aga ata

I

! PDKPS ISDLLAIGRG 3363 ccg gat aag cct tct ata tct gat ttg ctt get att ggg ege ggt j

! NDSYD ENKN GLLVLD 3408 aat gat tee tac gat gaa aat aaa aac ggc ttg ctt gtt etc gat t

! ECGTW FNTRSWNDKE 3453 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa

I

! RQPII DWFLHARKLG 3498 aga cag ccg att att gat tgg ttt eta cat get cgt aaa tta gga

I

! WDI IFLVQDLSIVDK 3543 tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa [

! QARSA LAEH VVYCRR 3588 cag gcg cgt tct gca tta get gaa cat gtt gtt tat tgt cgt cgt

I

! LDRITLPFVGTLYSL 3633 ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt

I

! ITGSKMPLPKLHVGV 3678 att act ggc teg aaa atg cct ctg cct aaa tta cat gtt ggc gtt

I

! VKYGDSQLSPTVERW 3723 gtt aaa tat ggc gat tct caa tta age cct act gtt gag cgt tgg

I

! LYTGKNLYNAYDTKQ 3768 ett tat act ggt aag aat ttg tat aac gca tat gat act aaa cag |

! AFSSNYDSGVY SYLT 3813 get ttt tct agt aat tat gat tee ggt gtt tat tct tat tta aeg i

! PYLSHGRYFKPLNLG 3858 cct tat tta tea cac ggt egg tat ttc aaa cca tta aat tta ggt i

! QKMKLTKIYLKKFSR 3903 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct ege !

! VLCLAIGFASAFTYS 3948 gtt ett tgt ett geg att gga ttt gca tea gca ttt aca tat agt i ·

! YITQPKPEVKKVVSQ 3993 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag

I

! TYDFDKFTIDS SQRL 4038 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ett t

! NLSYRYVFKDS KGKL

4083 aat eta age tat ege tat gtt ttc aag gat tct aag gga aaa TTA ! PacI

I

! INSDDLQKQGY SLTY

4128 ATT AAt age gac gat tta cag aag caa ggt tat tea etc aca tat ! PacI

I

! ilDLCTVSIKKGNSNE

! iv Ml K

4173 att gat tta tgt act gtt tee att aaa aaa ggt aat tea aAT Gaa ! Start IV i

! i I V K C N .End of I ! iv L3 L N5 V 17 N F V10 4218 att gtt aaa tgt aat TAA T TTT GTT ! IV continued..... 4243 ttc ttg atg ttt gtt tea tea tct tct ttt get cag gta att gaa atg 4291 aat aat teg cct ctg ege gat ttt gta act tgg tat tea aag caa tea 4339 ggc gaa tee gtt att gtt tct ccc gat gta aaa ggt act gtt act gta 4387 tat tea tct gac gtt aaa cct gaa aat eta ege aat ttc ttt att tct

4435 gtt tta cgt gcA aat aat ttt gat atg gtA ggt teT aAC cct tee atT 4483 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca 4531 tea tct gat aat cag gaa tat gat gat aat tee get cct tct ggt ggt 4579 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat 4627 aac gtt egg gca aag gat tta ata ega gtt gtc gaa ttg ttt gta aag 4675 tct aat act tct aaa tee tea aat gta tta tct att gac ggc tct aat 4723 eta tta gtt gtt agt geT cct aaa gat att tta gat aac ett cct caa 4771 ttc ett tcA act gtt gat ttg cca act gac cag ata ttg att gag ggt 4819 ttg ata ttt gag gtt cag caa ggt gat get tta gat ttt tea ttt get 4867 get ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac ege 4915 etc acc tct gtt tta tct tct get ggt ggt teg ttc ggt att ttt aat 4963 ggc gat gtt tta ggg eta tea gtt ege gca tta aag act aat age cat 5011 tea aaa ata ttg tct gtg cca cgt att ett aeg ett tea ggt cag aag 5059 ggt tct ate tct gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg ! MscI____ 5107 act ggt gaa tct gcc aat gta aat aat cca ttt cag acg att gag cgt 5155 caa aat gta ggt att tcc atg age gtt ttt cct gtt gca atg get ggc 5203 ggt aat att gtt ctg gat att acc age aag gcc gat agt ttg agt tct 5251 tct act cag gca agt gat gtt att act aat caa aga agt att get aca 5299 acg gtt aat ttg cgt gat gga cag act ett tta etc ggt ggc etc act 5347 gat tat aaa aac act tct caG gat tct ggc gta ccg ttc ctg tct aaa 5395 ate cct tta ate ggc etc ctg ttt age tec ege tct gat teT aac gag 5443 gaa age acg tta tac gtg etc gtc aaa gca acc ata gta ege gcc ctg

5491 TAG cggcgcatt ! End IV 5503 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 5563 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcGCCGGCt ttccccgtca ! NgoMI. 5623 agctctaaat cgggggctcc ctttagggtt ccgatttagt getttaegge acctcgaccc 5683 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt ! Drain---- 5743 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac ! DrdI.......... 5803 aacactcaac cctatctcgg getattettt tgatttataa gggattttgc cgatttcgga 5863 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5923 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga ! PvuII. BsmBI. 5983 aaaaccaccc tGGATCC AAGCTT ! BamHI Hindlll (1/2) ! Insert carrying bla gene 6006 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc

6043 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct ! BciVI 6103 gataaatget tcaataatat tgaaaaAGGA AGAgt ! RBS.?... ! Start bla gene 6138 ATG agt att caa cat ttc cgt gtc gcc ett att ccc ttt ttt geg gca ttt 6189 tgc ett cct gtt ttt get cac cca gaa acg ctg gtg aaa gta aaa gat get 6240 gaa gat cag ttg ggC gcA CTA GTg ggt tac ate gaa ctg gat etc aac age ! Spel. . . . ! ApaLI &amp; BssSI Removed 6291 ggt aag ate ett gag agt ttt ege ccc gaa gaa cgt ttt cca atg atg age 6342 act ttt aaa gtt ctg eta tgt GGC GeG Gta tta tec cgt att gac gcc ggg

6393 caa gaG CAA CTC GGT CGc cgC ATA cAC tat tct cag aat gac ttg gtt gAG ! Bcgl............ Seal 6444 TAC Tea cca gtc aca gaa aag cat ett acg gat ggc atg aca gta aga gaa ! Seal. 6495 tta tgc agt get gcc ata acc atg agt gat aac act geg gcc aac tta ett 6546 ctg aca aCG ATC Gga gga ccg aag gag eta acc get ttt ttg cac aac atg ! Pvul____ 6597 ggg gat cat gta act ege ett gat cgt tgg gaa ccg gag ctg aat gaa gcc 6648 ata cca aac gac gag cgt gac acc acg atg cct gta gca atg Gca aca acg 6699 tTG CGC Aaa eta tta act ggc gaa eta ett act eta get tec egg caa caa. ! FspI.... i 6750 tta ata gac tgg atg gag geg gat aaa gtt gca gga cca ett ctg ege teg 6801 GCC ett ccG GCt ggc tgg ttt att get gat aaa tct gga gcc ggt gag cgt ! Bgll.......... 6852 gGG TCT Cgc ggt ate att gca gca ctg ggg cca gat ggt aag ccc tec cgt ! Bsal.. . . 6903 ate gta gtt ate tac aeG ACg ggg aGT Cag gca act atg gat gaa ega aat ! Ahdl........... 6954 aga cag ate get gag ata ggt gcc tea ctg att aag cat tgg TAA ctgt ! stop 7003 cagaccaagt ttactcatat ataetttaga ttgatttaaa acttcatttt taatttaaaa 7063 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 7123 cgttccactg tacgtaagac cccc 7147 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct ! HindiII Sail.. ! (2/2) Hindi 7183 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt 1 ! Start of Fab-display cassette, the Fab DSR-A05, selected for ! binding to a protein antigen.

I 7233 CCTGAcG CTCGAG ! xBsu36I Xhol..

I ! PlacZ promoter is in the following block i 7246 cgcaacgc aattaatgtg agttagctca 7274 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 7324 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7374 tgattacgCC AagcttTGGa gccttttttt tggagatttt caac ! PflMI....... ! Hind3. (there are 3) ! Gene iii signal sequence: ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

! MKKLLFAIPLVVPFY 7418 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat

I ! 16 17 18 Start light chain (L20-.JK1)

! SHSAQDIQMTQSPA 7463 tct cac aGT GCA Caa oac ate cap ato acc cag tet cca gcc ! ApaLI... ! Sequence supplied by extender............

I

! T L S L 7505 acc ctg tct ttg .

I

! SPGERATLS CRAS QG 7517 tct cca ggg gaa aga gcc acc etc tcc tgc agg gcc agt cag Ggt

I

! VSSYLAWYQQKPGQA

7562 gtt age age tac tta gcc tgg tac cag cag aaa cct ggc cag get ; -! PRLLI YDAS SRATGI 7607 ccc agg etc etc ate tat gAt gca tcc aAc agg gcc act ggc ate |

! PARFSGSGPGTDFTL 7652 cca gCc agg ttc agt ggc agt ggg Cct ggg aca gac ttc act etc j

! TISSLEPEDFAVYYC 7697 acc ate age agC ctA gag cct gaa gat ttt gca gtT tat tac tgt |

! QQRSWHPWT FGQGTR

7742 cag cag CGt aAc tgg cat ccg tgg ACG TTC GGC CAA GGG ACC AAG

I

! VEIKRTVAAPSVFIF 7787 gtg gaa ate aaa ega act gtg gCT GCA Cca tct gtc ttc ate ttc ! Bsgl.... i

! PPSDEQLKS GTASVV 7832 ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg i

! CLLNN FYPR EAKV QW 7877 tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg

I

! KVDNALQSGNSQESV 7922 aag gtg gat aac gcc etc caa teg ggt aac tee cag gag agt gtc t

! TERDSKDSTYSLSST 7967 aca gag egg gac age aag gac age ace tac age etc age age ace j

! LTLSKADYEKHKVYA 8012 ctg aeG CTG AGC aaa gca gac tac gag aaa cac aaa gtc tac gcc ! EspI..... i

! CEVTHQG LSSPVTKS 8057 tgc gaa gtc acc cat cag ggc ctG AGC TCg ccc gtc aca aag age ! SacI.... i ! F N R G E C . . 8102 ttc aac agg gga gag tgt taa taa

I 8126 GGCGCG CCaattctat ttcaaGGAGA cagtcata ! AscI..... RBS2. i ! PelB signal sequence------(22 codons)-----> ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

! MKYLLPTAAAGLLLL 8160 atg aaa tac eta ttg cct aeg gca gcc get gga ttg tta tta etc

I ! ...PelB signal------------> Start VH, FR1-----------------> ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

! AAQPAMAEVQLLESG 8205 geG GCC cag ccG GCC atg gcc gaa gtt CAA TTG tta gag tet ggt ! Sfil............. Mfel. . . ! Ncol....

I ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

! GGLVQPGGSLRLSCA 8250 ggc ggt ett gtt cag cct ggt ggt tet tta cgt ett tet tgc get

I ! . . . FR1--------------------> CDR1--------------> FR2--------> ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

! ASGFTFSTYEMRWVR 8295 get TCC GGA ttc act ttc tet act tac gag atg cgt tgg gtt cgC ! BspEI.. BstXI.. . | ! FR2--------------------------------------> CDR2----------> ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

! QAPGKGLEWVSYIAP 8340 CAa get ccT GGt aaa ggt ttg gag tgg gtt tet tat ate get cct ! BstXI................ i ! ...CDR2---------------------------------------------> FR3----> ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

! SGGDTAYADSVKGRF 8385 tet ggt ggc gat act get tat get gac tee gtt aaa ggt ege ttc

I ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! TISRDNSKNTLYLQM 8430 act ate TCT AGA aac aac tet aaa aat act etc tac tta cag atg ! Xbal- . ! Supplied by extender-------------------------------

I ! -----------------------------------------FR3--------------> ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! NSLRAEDTAVYYCAR 8475 aac aaC TTA AGa act aaa aac act gca gtc tac tat tgt gcg agg ! AfIII. . . ! from extender---------------------------------> ! CDR3--------------------------------------------------- FR4 —> ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

! RLDGYISYYYGMDVW 8520 agg etc gat ggc tat att tcc tac tac tac ggt atg GAC GTC tgg ! Aatll.. 1 ! 136 137 138 139 140 141 142 143 144 145

! GQGTTVTV.SS 8565 ggc caa ggg acc acG GTC ACC gtc tea age ! BstEII... i ! CHI of IgGl---------->

I ASTKGPSVFPLAPSS 8595 gee tec acc aag ggc cca teg gtc ttc ccc ctg gca ccc tec tec 1

! KSTSGGTAALGCLVK 8640 aag age acc tet ggg ggc aca gcg gee ctg ggc tgc ctg gtc aag

I

i DYFPEPVTVSWNSGA 8685 gac tac ttc ccc gaa ccg gtg aeg gtg teg tgg aac tea ggc gee j

! LTSGVHTFPAVLQSS

8730 ctg acc age ggc gtc cac acc ttc ccg get gtc eta cag tCC TCA ! Bsu36I.... i

i G LYSLSSVV TVPSS S 8775 GGa etc tac tcc etc age age gta gtg acc gtg ccc tcc age age ! Bsu36I---- !

! LGTQTYICNVNHKPS 8820 ttg ggc acc cag acc tac ate tgc aac gtg aat cac aag ccc age

I

! NTKVDKKVE PKSCAA

8865 aac acc aag gtg gac aag aaa gtt gag ccc aaa tet tgt GCG GCC I Not I......

I

! AHHHHHHGAAEQKLI 8910 GCa cat cat cat cac cat cac ggg gee gca gaa caa aaa etc ate ! .. NotI.... H6 tag................. Myc-Tag........................

I

! SEEDLNGAAqASSA 8955 tea gaa gag gat ctg aat ggg gee gca tag GCT AGC tet get ! Myc-Tag.................... -- · Nhel. . . ! Amber

I ! Ill'stump i ! Domain 3 of III -------------------------------------------------------

I

! SG DFDYEKMA NANKG A 8997 agt ggc gac ttc gac tac gag aaa atg get aat gee aac aaa GGC GCC ! tcc tttttag acttggt !W.T. ! Kasl...(2/4) 1

! MTENADENAL QSDAK G 9045 atG ACT GAG AAC GCT GAC GAG aat get ttg caa age gat gee aag ggt ! catctacgcag tet c t a c !W.T. |

! KLDSVATDYGAAIDGF 9093 aag tta gac age gTC GCG Acc gac tat GGC GCC gee ATC GAc ggc ttt ! a c t t tet tttcttt ttc ! W. T. ! Nrul... . Kasl... {3/4)

I

! IGDVSGLANGNGATGD 9141 ate ggc gat gtc agt ggt tTG GCC Aac ggc aac gga gee acc gga gac ! t t c t tee cctttttttttt !W.T. ! MscI....{3/3)

I

! FAGS NS QMAQV GDGDN 9189 ttc GCA GGT teG AAT TCt cag atg geC CAG GTT GGA GAT GGg gac aac ! ttet ca tactcttt !W.T. ! BspMI.. (2/2) XemI................ ! EcoRI... i

! S PLMNN FRQYLPSLPQ 9237 agt ccg ett atg aac aac ttt aga cag tac ett ccg tet ett ccg cag ! tea tta t t cct a tta t c c t a !W.T. |

! SVECRP FVFSAGKPYE 9285 agt gtc gag tgc cgt cca ttc gtt ttc tet gee ggc aag cct tac gag ! teg tatettet age t t a a t a !W.T. i

! FSIDCDKINLFR

9333 ttc aGC Ate gac TGC gat aag ate aat ett ttC CGC ! t tet t t t c a a eta c t !W.T. ! BstAPI........ SacII... ! End Domain 3 i

! GVFAFLLYVATFMYVF 9369 GGc gtt ttc get ttc ttg eta tac gtc get act ttc atg tac gtt ttc ! tctgtcttattcct tat !W.T. ! start transmembrane segment t

! STFANIL RNKES 9417 aGC ACT TTC GCC AAT ATT TTA Cgc aac aaa gaa age ! tet gttcacg ttgg tet !W.T. ! Intracellular anchor. i m i . .

9453 tag tga tet CCT AGG ! AvrII.. j

9468 aag ccc gee taa tga geg ggc ttt ttt ttt ct ggt ! I Trp terminator I

I ! End Fab cassette

I 9503 ATGCAT CCTGAGG ccgat actgtcgtcg tcccctcaaa ctggcagatg ! Nsil.. Bsu36I.(3/3) 9551 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg 9611 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc 9671 tggctacagg aaggccagac gegaattatt tttgatggcg ttcctattgg ttaaaaaatg

9731 agctgattta acaaaaattt aaTgegaatt ttaacaaaat attaacgttt acaATTTAAA ! Swal... 9791 Tatttgetta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat 9850 ATG att gac atg eta gtt tta ega tta ccg ttc ate gat tet ett gtt tgc

! Start gene II 9901 tcc aga etc tea ggc aat gac ctg ata gee ttt gtA GAT CTc tea aaa ata ! Bglll... 9952 get ace etc tec ggc atT aat tta tea get aga aeg gtt gaa tat cat-att 10003 gat ggt gat ttg act gtc tec ggc ett tet cac cct ttt gaa tet tta cct 10054 aca cat tac tea ggc att gca ttt aaa ata tat gag gg* tet aaa aat ttt 10105 tat cct tgc gtt gaa ata aag get tet ccc gca aaa gta tta cag ggt cat 10156 aat gtt ttt ggt aca ace gat tta get tta tgc tet gag get tta ttg ett 10207 aat ttt get aat tet ttg cct tgc ctg tat gat tta ttg gat gtt ! ! gene II continues I------------------------End 0f Table------------------------------- ! Table 37: DNA seq of w.t. M13 gene iii i ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

! fMKKLLFAI PLVV P FY 1579 gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat ! Signal sequence............................................ | ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

! SHSAETVE SCLAKPH 1624 tct cac tcc get gaa act gtt gaa agt tgt tta gca aaa ccc cat ! Signal sequencer Domain 1--------------------------------------- t ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

! TENS FTNVWKDDKTL 1669 aca gaa aat tea ttt act aac gtc tgg aaa gac gac aaa act tta ! Domain 1--------------------------------------------------- i ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

! DRYANYEGCLWNATG 1714 gat cgt tac get aac tat gag ggt tgt ctg tgG AAT GCt aca ggc ! BsmI.... ! Domain 1--------------------------------------------------- i ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

! VVVCTGDE TQCY GTW 1759 gtt gta gtt tgt act ggt gac gaa act cag tgt tac ggt aca tgg ! Domain 1--------------------------------------------------- i ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

! VPI GLAI P ENEGGGS 1804 gtt cct att ggg ett get ate cct gaa aat gag ggt ggt ggc tct ! Domain 1------------------------------> Linker 1----------- i ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

! EGGGSEGGGSEGGGT 1849 gag ggt ggc ggt tct gag ggt ggc ggt tct gag ggt ggc ggt act ! Linker 1--------------------------------------------------> 1 ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

! KPPEYGDT PI PGYTY 1894 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat ! Domain 2--------------------------------------------------- ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

! INPLDGTY PPGT EQN 1939 ate aac cct etc gac ggc act taT CCG CCt ggt act gag caa aac ! Ecil---- ! Domain 2---------------------------------------------------

J ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150

! PANPNPSLEESQ PLN 1984 ccc get aat cct aat cct tct ett GAG GAG tct cag cct ett aat ! BseRI.. ! Domain 2----------------------------------------------------

I ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

! TFMFQNNRFRNRQGA 2029 act ttc atg ttt cag aat aat agg ttc ega aat agg cag ggg gca ! Domain 2---------------------------------------------------

I ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

I LTVYTGTVTQGTDPV 2074 tta act gtt tat acg ggc act gtt act caa ggc act gac ccc gtt ! Domain 2-----------------------------------------------------

I ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

! KTYYQYTPVSSKAMY 2119 aaa act tat tac cag tac act cct gta tea tea aaa gee atg tat ! Domain 2---------------------------------------------------

I i 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 I daywngkfrdcafhs 2164 gac get tac tgg aac ggt aaa ttC AGa gaC TGc get ttc cat tet ! AlwNI....... ! Domain 2---------------------------------------------------

I i 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225

! GFNEDPFVCEYQGQS 2209 ggc ttt aat gaG GAT CCa ttc gtt tgt gaa tat caa ggc caa teg ! BamHI... ! Domain 2--------------------------------------------------- ! ! 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

i SDLPQPPVNAGGGSG 2254 tet gac ctg cct caa cct cct gtc aat get ggc ggc ggc tet ggt ! Domain 2------------------------------> Linker 2 ! ! 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 1 ggsgggsegggsegg 2299 ggt ggt tet ggt ggc ggc tet gag ggt ggt ggc tet gag ggt ggc ! Linker 2---------------------------------------------------

J ! 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 I gsegggsegggsggg 2344 ggt tet gag ggt ggc ggc tet gag gga ggc ggt tee ggt ggt ggc ! Linker 2---------------------------------------------------

I ! 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285

! SGSGDFDYEKMANAN 2389 tet ggt tee ggt gat ttt gat tat gaa aag atg gca aac get aat !Linker 2> Domain 3-------------------------------------------

I ! 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

! KGAMTENADENALQS 2434 aag ggg get atg acc gaa aat gee gat gaa aac geg eta cag tet ! Domain 3---------------------------------------------------

I ! 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

! DAKGKLDSVAT DYGA 2479 gac get aaa ggc aaa ett gat tet gtc get act gat tac ggt get ! Domain 3---------------------------------------------------

I ! 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330

! A I DGFIGDVSGLANG 2524 get ate gat ggt ttc att ggt gac gtt tee ggc ett get aat ggt ! Domain 3---------------------------------------------------

I ! 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345

! NGA TGDFAGSNSQMA 2569 aat ggt get act ggt gat ttt get ggc tet aat tee caa atg get ! Domain 3--------------------------------------------------- j ! 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

! QVGDGDNSPLMNNFR 2614 caa gtc ggt gac ggt gat aat tea cct tta atg aat aat ttc cgt ! Domain 3---------------------------------------------------

I ! 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375

! QYLPSLPQSV ECRPF 2659 caa tat tta cct tee etc cct caa teg gtt gaa tgt ege cct ttt ! Domain 3--------------------------------------------------- ] t 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

! VFSAGKPYEFTIDCD 2704 gtc ttt age get ggt aaa cca tat gaa ttt tet att gat tgt gac ! Domain 3---------------------------------------------------

J ! 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405

1 KINLFRGVFA FLLYV 2749 aaa ata aac tta ttc cgt ggt gtc ttt geg ttt ett tta tat gtt ! Domain 3--------------> Transmembrane segment--------------

I ! 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

! ATFMYVFSTFANILR

2794 gee acc ttt atg tat gta ttt tet aeg ttt get aac ata ctg cgt ! Transmembrane segment---------------------------------> ICA i ! 421 422 423 424 425 ! N K E S . 2839 aat aag gag tet taa ! 2853 ! ICA-----------> ICA = intracellular anchor

I i------------------End of Table-----------------------------------------

Table 38: Whole mature III anchor M13-III

derived anchor with recoded DNA

I ! 12 3

! AAA 1 GCG gcc gca 1 Notl...... ! 4 5 6 7 8 9 10 11 12 13 14 15 16 17

I HHHHHHGAAEQKLI 10 cat cat .cat cac cat cac ggg gcc gca gaa caa aaa etc ate * ! 18 19 20 21 22 23 24 25 26 27 28 29

! SEEDLNGAA.AS

52 tea gaa gag gat ctg aat ggg gcc gca Tag GCT AGC i Nhel. . .

I ! 30 31 32 33 34 35 36 37 38 39

! DINDDRM AST 88 GAT ATC aac aat aat cat atq get tet act ! (ON_G37bot) [RC] 5'-c aac aat oat cat atq geG CAt Get gcc gag aca g-3' ! EcoRV.. ! Enterokinase cleavage site. 1 ! Start mature III (recoded) Domain 1----> ! 40 41 42 43

! A E T V 118 |gcCIgaGIacA|gtC| ! t a t t ! W.T.

I ! 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

i ESCLAKPHTENS FTN 130 IgaaITCCItgCICTGIGCCIAaGIccTIcaC|acT|gaGIaat | AGTIttC|aCA|Aat| ! agt t t a a a c t a a tea t t c ! W.T. ! MscI.... I e ! 59 60 61 62 63 64 *65 66 67 68 69 70 71 72 73

i VWKDDKTLDRYANYE 17 5 IgtgITGGIaaGIgaT|gaTIaaGIacC|CtT|gAT|CGA| TaT|gcC|aaT|taC|gaA | ! c accatta tetetg! W.T. i BspDI... | ! 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

! GCLWNATGVVVCTGD 220 |ggCItgCITtAItggIaatIgeCI ACC IGGC|GtCIgtTIgtC|TGCIACG|ggC|gaT| ! tteg ta tattttc! W.T. ! SgrAI...... Bsgl....

I ! 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

! ETQCYGTWVPIG LAI 2 65 )gaGIacAIcaA|tgCItaTIggCIACG|TGg|gtGIccGI atAI gGC|TTA|GCC|atA | ! atgteta tttgette! W.T. ! Pmll---- BlpI..... | ! Domain 1-----> Linker 1----------------> ! 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

! PENEGGGSEGGG SEG 310 |ccGIgaG|aaC|gaA|ggCIggC|ggTIAGCIgaAIggCIggT | ggC|AGC|gaA|ggC| ! t a t g t t c tet g t c t tet g t ! W.T.

I ! Linker 1----------------------> Domain 2---------------> ! 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

! GGSEGGGTKPPEYGD 355 IggTIGGAITCCIgaA|ggA|ggTIggA|acCIaaG[ccGIccG|gaA11aT | ggC I gaC I ! cttgtct t at tg ctt! W.T. ! BamHI..(2/2) ! ! 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148

! TPIPGYTYINPLDGT 400 I acT | ccG | atA I CCT | GGT | taC | acC 11 aC I atT | aaT [ ccG i TtA I gaT | ggA I acC I ! attgctttcctcccct! W.T. ! SexAI.... f ! 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163

i yppGTEQNPANPN PS

4 4 5 |taCIccTIccGIggCIacC|gaA|caG|aaT|ccT|gcC|aaC|ccGIaaCI ccA IAGCI < TGtttgaccttttt tct ! W.T. t Hindlll...

I

! 164 165 166 167 168 169 170 171 172 173 174 175 176 177 17B

! LEESQPLNTFMFQNN

4 90 | TTA | gaA I gaA | AGC I caA| ccG I TtA I aaC I acC | ttT | atgl ttC I caAl aaCI aaC I ! c t G G tct g tct t t C t g t t!W.T. ! Hindlll. 1 ! 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193

! RFRNRQGALTVYTGT 535 I CgT | ttT | AgG | aaC | CgT | caA I gGT IGCT | CtT | acC I gTG I TAC I AcT | ggA I acC I ! ag c c a tag g g ata t t t g c t! W.T. ! HgiAI... BsrGI... ! 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

! VTQGTDPVKTYY QYT

580 IgtCIacCIcaGIGGTI ACC|gaT|ccT|gtC|aaG|acC|taC|taT|caA | taT I acCI ! ttactcctattcgct! W.T. ! Kpnl... r i 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223

! PVSSKAMYDAYWNGK 625 IccG|gtCITCGIAGtIaaGIgcT|atgItaCIgaTIgcCItaT|tggIaaT | ggC IaaG| ! t a a tea ac tctc eta! W.T. ! Bsal.... ! Xhol---- 1 ! 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

! FRDCAFHSGFNEDPF 670 IttTICgT|gaT|tgT|gcC|ttT|caC|AGC|ggT|ttCIaaCIgaaIgacICCtIttT| ! CAaCctct tct C t t G T a c!W.T.

I ! 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253

! VCEYQGQSSDLPQ PP 715 IgtCItgCIgaGItaC|caGIggT|caG|AGT|AGC|gaTITtA|ccG|caGI ccA I CCGI ! t t a t a c a teg tct ccg t a t t!W.T. ! DrdI..... Agel.....

J ! Domain 2--------> Linker 2---------------------> ! 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268

! VNAGGGSGGGSGGGS

7 60 | GTT | AAC | geG | ggT I ggT I ggT! AGC I ggC | ggA I ggC I AGC I ggC I ggT | ggT I AGC I ! c t t c c c tct t t t tct tee tct ! W.T. ! AgeI..... ! Hpal. . . ! Hindi.

I ! Linker 2----------------------------------------------- Domain 3—> ! 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283

j EGGGSEGGGSGGGSG 805 |gaA|ggCIggAIggTIAGC|gaAIggAIggTIggC1AGC|ggA|ggCIggTIAGCIggCI I g t t c tct g t c t tct g t c tct t ! W.T. | I ------------Domain 3-------------------> ! 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298

I SGDFDYEKMANANKG 850 |AGTIggC|gaclttcIgacI tad gag IaaaIatg|get|aat|gee|aac|aaa|GGC| i tee tttttag aettgg! W.T. i KasI.... ! 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 I am tenadenal qsda 8 95 |GCCIatgI act I gag IaacI get IgacIgaGIAAT|GCA|ctg|caa | agt I gat IgCCI i t catctacgag tct c t ! W.T. ! KasI.... BsmI.... Styl...

I ! 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328

! KG KLDSVATDY GAAI 940 |AAGIGGtIaagIttaIgacI age IgTCiGCctAcaIgacltat|ggT|GCt|gee I ate| ! a c a c t t tct t t t c t ! W.T. ! Styl...... PflFI...... t ! 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343

! DGFIGDVSGLANGNG 985 IgacIggc i tttI ate Iggc|gat|gtc|agt|ggtIctg j get IaacIggcIaacIgga| ! t t c t t c ttcc cct t t t t ! W.T.

I ! 344 345 346 347 348 349 350 351 352 353

! ATGDFAGSNS 1030 I gee I ace IggaIgacIttc|GCA|GGT|teG|AAT|TCt| ! ttttttct c ! W.T. ! BstBI... ! EcoRI... ! BspMI..

J ! 354 355 356 357 358 359 360 361 362 363

! QMAQVGDGDN 1060 cag atg geC CAG GTT GGA GAT GGg gac aac ! a tactctttiW.T. ! XemI................ | ! 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379

! SP LMNNFRQYL PSLPQ 1090 agt ccg ett atg aac aac ttt aga cag tac ett ccg tct ett ccg cag ! tea tta t t cct a tta t c c t a! W.T.

I ! 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395

! SVECRPFVFSAGKPYE 1138 agt gtc gag tgc cgt cca ttc gtt ttc tct gee ggc aag cct tac gag ! teg t atettet age t t aat a ! W.T.

I ! Domain 3--------------------------------------> ! 396 397 398 399 400 401 402 403 404 405 406 407

! FSIDCDKINLFR 1186 f.z aGC Ate gac TGC gat aag ate aat ett ttC CGC ! t tct ttt caa eta t ! BstAPI........ SacII. . .

I i transmembrane segment-------------> i 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423

I GVFAFLLYVATFMYVF 1222 GGc gtt ttc get ttc ttg eta tac gtc get act ttc atg tac gtt ttc I t c t g t c t t attcct tat! W.T.

I i 424 425 426 427 428 429 430 431 432 433 434 435

! STFA.NIL RNKES 1270 aGC ACT TTC GCC AAT ATT TTA Cgc aac aaa gaa age ! tet g t t c a c g t t g g tet ! W.T. I Intracellular anchor. !

1306 tag tga tet CCT AGG ! AvrII. . i

1321 aag ccc gee taa tga geg ggc ttt ttt ttt ct ggt ! | Trp terminator I i ! End Fab cassette -----------------------------End of Table-------------------------

Table 39: ONs to make deletions in III ! ONs for use with NheI

I

N (ON_G29bot) 5'-c gTT gAT ATc gcT Age cTA Tgc-3' ! 22 “ . ! this is the reverse complement of 5'-gca tag get age gat ate aac g-3' j Nhel... scab......... (ON_G104top) 5'-gIataIggcIttaIgcTIaGC|ccg|gag|aacIgaaIgg-3' ! 30 i Scab..........Nhel... 104 105 106 107 108 (ON_G236top) 5 · -c I ttt I cac i age I ggt | ttc I GCT | AGC I gac I cct I ttt | gtc | tgc-3 ’ ! 37 i Nhel. . . 236 237 238 239 240 (ON G236tCS) 5'-cItttIcacI age Iggt|ttc|GCT|AGCIgacIcctIttt|gtc | Agc-i “ Nhel... 236 237 238 239 240 gag|tacIcag|ggt|c-3 ' ! 50 ! ONs for use with SphI G CAT Gc (ON X37bot) 5'-gAc TgT cTc ggc Age ATg ege cAT Acg ATc ATc gTT g-3' ! 37 “ .

1 N DDRMAHA ! (ON_X37bot) = [RC] 5'-c aac gat gat cgt atg qcG CAt Get gcc gag aca gtc-3' i SphI....Scab........... (0N_X104top) 5'-g|gtG ccg|ataIggcIttGI CAT|GCaiccg|gag Iaac|gaa|gg-3' ! 36 t Scab.................SphI____ 104 105 106 107 108 (ON_X236top) 5 ' -c I ttt I cac I age | g-gt i ttG | CaT | gCa | gac I cct | ttt I gtc | tgc-3 ' ! 37 - ! SphI____ 236 237 238 239 240 (ON X236tCS) 5'-c|ttt|cac|age|ggtIttGICaT|gCaIgacIcct I tttIgtcIAge-i - Nhel... 236 237 238 239 240 gag|tac|cag|ggtIc-3 ' ! 50

Table 40: Phage titers and enrichments of a selections with a DY3F31—based human Fab library

Table 41: Frequency of ELISA positives in DY3F31-based Fab libraries

Claims (18)

1. A vector comprising: (i) a nucleic sequence encoding an antibody variable region linked to a fragment of a wild-type pill anchor, which does not mediate infection of phage particles; and (ii) a wild-type gene III, wherein the nucleic acid sequence encoding the fragment of the wild-type pill anchor differs from the wild-type gene III.

2. The vector of claim 1, wherein the nucleic acid sequence encoding the fragment of the wild-type pill anchor differs from the wild-type gene III to an extent sufficient to reduce homologous recombination between the nucleic acid encoding the fragment of the wild-type PHI anchor and the wild-type gene III.

3. The vector of claim 2, wherein all codons in the nucleic acid sequence encoding the fragment of the wild-type pill anchor have at least one base change introduced per codon compared to the wild-type gene III.

4. The vector of any one of claims 1 to 3, wherein the nucleic acid sequence set forth in (i) encodes a Fab.

5. The vector of any one of claims 1 to 3, wherein the nucleic acid sequence set forth in (i) encodes a heavy chain VHCHl.

6. The vector of claim 5, wherein the heavy chain VHCHl is linked to trpllI.

7. The vector of any one of claims 1 to 3, wherein the nucleic acid sequence set forth in (i) encodes a light chain VLCL.

8. The vector of claim 5 or claim 6, wherein the nucleic acid sequence set forth in (i) further encodes a light chain VLCL.

9. The vector of claim 7 or claim 8, wherein the light chain VLCL is linked to trpIII.

10. The vector of any one of claims 1 to 3, wherein the nucleic acid sequence set forth in (i) encodes a scFv.

11. The vector of claim 10, wherein the scFv is a VL-VH.

12. The vector of claim 10, wherein the scFv is a VH-VL.

13. The vector of any one of claims 1 to 12, wherein the nucleic acid sequence encoding the antibody variable region further comprises an inducible promoter.

14. The vector of claim 13, wherein the inducible promoter regulates expression of the nucleic acid sequence encoding the antibody variable region.

15. The vector of any one of claims 1 to 14, wherein the nucleic acid sequence encoding the antibody variable region further comprises an amber stop codon.

16. The vector of claim 15, wherein the amber stop codon is located between the antibody variable region and the fragment of the wild-type pill anchor.

17. The vector of any one of claims 1 to 16, wherein the vector is phage or phagemid.

18. The vector of any one of claims 1 to 17, wherein the vector further comprises a wild-type gene VIII. Date: 18 May 2016