# Patent

## Title
Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries

## Abstract

A library containing a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the family, the displayed peptides, polypeptides or proteins being encoded by DNA sequences containing sequences encoding a heavy chain CDR region is described.

## Background
This application is a continuation-in-part of U.S. patent application Ser. No. 09/837,306, filed on Apr. 17, 2001, which claims the benefit of U.S. provisional application 60/198,069, filed on Apr. 17, 2000. All of the earlier applications are specifically incorporated by reference herein.


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.
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 B1, 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
It is an object of this invention to 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.
It is another object of this invention to 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 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.

    
    


It is a further object of this invention to provide an alternative 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 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.

    
    


In an alternative embodiment of this object of the invention, the restriction endonuclease recognition site is not initially located in the double-stranded part of the oligonucleotide. Instead, it is part of an amplification primer, which primer is complementary to the double-stranded 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.
It is another object of the present invention to 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 double-stranded region of the partially double-stranded oligonucleotide.
    
    


As before, in this object of the invention, 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.
It is another object of this invention to prepare 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.
It is an object of this invention to screen those libraries to identify useful peptides, polypeptides and proteins and to use those substances in human therapy.
Additional objects of the invention are reflected in claims 1-116. Each of these claims is specifically incorporated by reference in this specification.



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. The T15 oligonucleotide is shown in SEQ ID NO: 622.
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 DNA from Example 2.
FIG. 6 depicts gel analysis of extender-cleaved kappa DNA from Example 2.
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 (SEQ ID NO:636) and CDR2 (SEQ ID NO:637) synthetic diversity.
FIG. 11 is a schematic 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. The PCRpr oligonucleotide is shown in SEQ ID NO: 605 while the Bridge oligonucleotide and encoded peptide are shown in SEQ ID NOS 606-607, respectively.
FIG. 19 is a schematic of a process for incorporating fixed FR1 residues in an antibody kappa sequence (see SEQ ID NOS 608-611, respectively, in order of appearance).
FIG. 20 is a schematic of a process for incorporating fixed FR1 residues in an antibody heavy chain sequence. The PCRpr oligonucleotide is shown in SEQ ID NO: 612. The Bridge oligonucleotides are shown in SEQ ID NOS 613 & 615, respectively, in order of appearance, while the encoded peptides are shown in SEQ ID NOS 614 & 616, respectively, in order of appearance.



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



URE
Universal restriction




endonuclease



Functionally
Two sequences are sufficiently



complementary
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., at non-specific sites other than the specific cleavage site that is characteristic of the restriction endonuclease. The skilled worker will recognize that such non-specific, 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 double-stranded oligonucleotide duplexing with the single-stranded 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 single-stranded 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 immunoglobin 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. Pat. No. 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., H J 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 amide-forming 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 PCR(TM)), 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 double-stranded (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 AscI. 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., & 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. 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 single-stranded form.
Enzymes shown in Table 2 that cut many of the heavy chain FR3 germline genes at a single position include: MaeIII(24@4), Tsp45I(21@4), HphI(44@5), BsaJI(23@65), AluI(23@47), BlpI(21@48), DdeI(29@58), BglII(10@61), MslI(44@72), BsiEI(23@74), EaeI(23@74), EagI(23@74), HaeIII(25@75), Bst4CI(51@86), HpyCH4III(51@86), HinfI(38@2), MlyI(18@2), PleI(18@2), MnlI(31@67), HpyCH4V(21@44), BsmAI(16@11), BpmI(19@12), XmnI(12@30), and SacI(11@51). (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 TaaI or HpyCH4III), BlpI, HpyCH4V, and MslI. Because ACNGT (the restriction endonuclease recognition site for Bst4CI, TaaI, and HpyCH4III) 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 TaaI 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 (tgy) 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.1. 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 FR1s. The preferred sites are BsgI(GTGCAG;39@4), BsoFI(GCngc;43@6,11@9,2@3,1@12), TseI(Gcwgc;43@6,11@9,2@3,1@12), MspA1I(CMGckg;46@7,2@1), PvuII(CAGctg;46@7,2@1), AluI(AGct;48@82@2), DdeI(Ctnag;22@52,9@48), HphI(tcacc;22@80), BssKI(Nccngg;35@39,2@40), BsaJI(Ccnngg;32@40,2@41), BstNI(CCwgg;33@40), ScrFI(CCngg;35@40,2@41), Eco0109I(RGgnccy;22@46, 11@43), Sau96I(Ggncc;23@47,11@44), AvaII(Ggwcc;23@47,4@44), PpuMI(RGgwccy;22@46,4@43), BsmFI(gtccc;20@48), HinfI(Gantc;34@16,21@56,21@77), TfiI(21@77), MlyI(GAGTC;34@16), MlyI(gactc;21@56), and AlwNI(CAGnnnctg;22@68). The more preferred sites are MspAI and PvuII. MspAI 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 germline 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. HinfI and DdeI 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 single-stranded 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 double-stranded 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 double-stranded 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 single-stranded 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, 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 available at Table 12 B.
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 single-stranded 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 FokI.
When the preferred FokI 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 FokI 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 FokI 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 FokI restriction endonuclease recognition site containing segments. In the preferred embodiment, the stem (containing the FokI site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these “lollipop” URE 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 FokI 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 sequences 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 interfere 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 TaaI.
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 AflII-site as well as the XbaI site can be added. This bottom strand portion of the partially-duplexed adaptor, H43.XAExt, incorporates both XbaI and AflII-sites. The top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments opposite the XbaI and AflII-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt. H43AExt contains only the AflII-site and is to be used with the top strands H43.ABr1 and H43.ABr2 (which have intentional alterations to destroy the AflII-site).
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 single-stranded 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, SfiI, NotI, AflII, ApaLI, and AscI are very suitable. SfiI and NotI are preferably used in pCES1 to insert the heavy-chain display segment. ApaLI and AscI are preferably used in pCES1 to insert the light-chain display segment.
BstEII-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/79 (68%) of heavy-chain genes contain a BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII-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 CH1.
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 double-stranded adapter (e.g., XbaI and AflII (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 (kaBR01UR, 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_LamEx133) and four bridge oligonucleotides (ON_LamB1-133, ON_LamB2-133, ON_LamB3-133, and ON_LamB4-133) 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_Lam133PCR 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, 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 double-stranded 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, NcoI, XbaI, AflII, BstEII, ApaI, and NotI sites. The light chains are preferably moved into the library as ApaLI-AscI fragments. The heavy chains are preferably moved into the library as SfiI-NotI 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., pCES1) 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 (“IIIstump”) and a second possible anchor being the full length III mature protein.
The IIIstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. III 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. iii and possibly viii genes are present, the w.t. proteins are also present. The display cassette is transcribed from a regulatable promoter (e.g., PLacZ). 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, SS1 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, CH1 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.
XL1-Blue MRF′ satisfies most of these preferences, but does not grow well at 30° C. XL1-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+. XL1-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 Arthritis 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 GeneRAacerTM 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 in the GeneRAacerTM 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 CH1 chain of IgM [HuCmFOR] and a backward primer based on the ligated synthetic adaptor sequence [5′A] 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. M1-2 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 PACE material with biotin attached to 5′-end of upper strand was immobilized as in Example 1 on 200 microliters (μL) of Seradyn magnetic beads. The lower strand was removed by washing the DNA with 2 aliquots 200 μL 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 μL 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 μL 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 mM 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 kapPCRt1 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 pCES1.
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 BsmA1 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 3-23 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—TOPFR1A, BOTFR1B, BOTFR2, BOTFR3, F06, BoTFR4, ON-vgC1, and ON-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 2.5 uM in a 20 ul PCR reaction. The PCR mixture contained 200 uM dNTPs, 2.5 mM MgCl2, 0.02 U Pfu TurboTM DNA Polymerase, 1 U Qiagen HotStart Taq DNA Polymerase, and 1× Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 30 s.
The assembled 3-23 VH DNA sequence was then amplified, using 2.5 ul of a 10-fold dilution from the initial PCR in 100 ul PCR reaction. The PCR reaction contained 200 uM dNTPs, 2.5 mM MgCl2, 0.02 U Pfu TurboTM DNA Polymerase, 1 U Qiagen HotStart Taq DNA Polymerase, 1× Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of 1 uM. The PCR program consisted of 23 cycles at 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 60 s. The 3-23 VH DNA sequence was digested and cloned into pCES1 (phagemid vector) using the SfiI and BstEII restriction endonuclease sites. All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, Mass. and used as per the manufacturer's instructions.
Stuffer sequences (shown in Table 28 and Table 29) were introduced into pCES1 to replace CDR1/CDR2 sequences (900 bases between BspEI and XbaI 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 over-represented 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, BclI, XcmI, MluI, PvuII, HpaI, and HincII, 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-vgC1, ON_Br12, ON_CD2Xba, and ON-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 2.5 uM in a 40 ul PCR reaction. Two of the 4 oligos contained variegated sequences positioned at the CDR1 and the CDR2. The PCR mixture contained 200 uM dNTPs, 2.5 U Pwo DNA Polymerase (Roche), and 1×Pwo PCR buffer with 2 mM MgSO4. The PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s. This assembled CDR1/2 DNA sequence was amplified, using 2.5 ul of the mixture in 100 ul PCR reaction. The PCR reaction contained 200 uM dNTPs, 2.5 U Pwo DNA Polymerase, 1×Pwo PCR Buffer with 2 mM MgSO4 and 2 outside primers at a concentration of 1 uM. The PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s. These variegated sequences were digested and cloned into the 3-23 VH framework in place of the CDR1/2 stuffer.
We obtained approximately 7×107 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 BspE1 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. 12A 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_Lam1aB7, 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 16° 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 equimolar 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 ApaL1 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 Potassium Acetate, 20 mM Tris-Acetate, 10 mM Magnesium 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 SfiI and NotI, 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 NotI 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 XbaI 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 SfiI 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) CH1,
        n) A NotI RERS,
        o) A His6 tag (SEQ ID NO: 12),
        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 (L1)::Domain 2 (D2)::Linker 2 (L2)::Domain 3 (D3)::Transmembrane Segment (TM)::Intracellular anchor (IC) (see Table 38).
The pIII anchor (also known as trpIII) preferably consists of D2::L2::D3::TM::IC. Another embodiment for the pIII 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 pIII 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 NotI site, His6 tag (SEQ ID NO: 12), 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. III 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 1 and 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., agc), 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., agc), 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 (Biotechniques 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 NheI site before the DINDDRMA (residues 29-36 of SEQ ID NO: 594) recombinant enterokinase cleavage site (rEKCS). If NheI 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-affinity antibodies. One could mutagenize this sequence so that the NheI 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 (R1-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 ApaL1-Not1 restriction sites.
DNA from the DY3F31 phage vector was pretreated with ATP dependent DNAse to remove chromosomal DNA and then digested with ApaL1 and Not1. An extra digestion with AscI was performed in between to prevent self-ligation of the vector. The ApaL1/NotI Fab fragment from the preselected libraries was subsequently ligated to the vector DNA and transformed into competent XL1-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 μl of electroporation-competent cells (electroporation conditions: one shock of 1700 V, 1 hour recovery of cells in rich SOC medium, plating on ampicillin-containing agar plates).
This transformation resulted in a library size of 1.6×106 for R1-ox in DY3F31 and 2.1×106 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. R1-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 μg/ml AMP to an OD600 of approximately 0.5, and then grown overnight in 2TY-50 μg/ml AMP+/−1 mM 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 μL of beads were blocked for each depletion and subsequent selection set.
Per selection, 6.25 μL 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, 2×1012 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 μL 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 μL 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 μL of depletion beads.
A small sample of each depleted library pool was taken for tittering. 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 5×0.500 mL PBST and then 2× with PBS. Phage still bound to beads after the washing were eluted once with 0.250 mL of competitive ligand ( ̃1 μμM) 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 tittering. 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 2×YT and an aliquot (e.g., 1 mL with 1. E 10/mL) of XL1-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 (243×243×18 mm, Nalge Nunc) containing 2×YT, 1 mM 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 10 min, keep as elution
            B. Bead infect. amp. elution; elute w/TEA 10 min, toss as wash elute w/cell infection, keep as elution

Mid-log Prep of XL1 Blue MRF′ Cells for Infection

(Based on Barbas et al. Phage Display Manual Procedure)

        
        
    
    


Culture XL1 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 2×250 mL centrifuge bottles, centrifuged at 600 g for 15 min at 4° C. Supernatant was poured off. Residual liquid was removed with a pipette.
The pellets were gently resuspended (not pipetting up and down) in the original volume of 1× 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 2×250 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 2×YT medium and 2.5 mL of cells were added. Beads were brought up to 3 mL with 2×YT and 1 mL of cells were added. Incubated at 37° C. for 30 min. The cells were plated on 2×YT, 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 & 4).
The process for incorporating fixed FR1 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 FR1 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 FR1 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 FR1 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 XbaI.
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.







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 atc agc aca gcc tac atg


 


!
81  82  82a 82b 82c 83  84  85  86  87  88  89  90  91  92



gag ctg agc agg ctg aga tct gac gac acg gcc gtg tat tac tgt


 


!
93  94  95



gcg aga ga ! 1-02# 1 (SEQ ID NO: 34)


 



aga gtc acc att acc agg gac aca tcc gcg agc aca gcc tac atg


 



gag ctg agc agc ctg aga tct gaa gac acg gct gtg tat tac tgt


 



gcg aga ga ! 1-03# 2 (SEQ ID NO: 35)


 



aga gtc acc atg acc agg aac acc tcc ata agc aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aga gg ! 1-08# 3 (SEQ ID NO: 36)


 



aga gtc acc atg acc aca gac aca tcc acg agc aca gcc tac atg


 



gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 1-18# 4 (SEQ ID NO: 37)


 



aga gtc acc atg acc gag gac aca tct aca gac aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aca ga ! 1-24# 5 (SEQ ID NO: 38)


 



aga gtc acc att acc agg gac agg tct atg agc aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac aca gcc atg tat tac tgt


 



gca aga ta ! 1-45# 6 (SEQ ID NO: 39)


 



aga gtc acc atg acc agg gac acg tcc acg agc aca gtc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 1-46# 7 (SEQ ID NO: 40)


 



aga gtc acc att acc agg gac atg tcc aca agc aca gcc tac atg


 



gag ctg agc agc ctg aga tcc gag gac acg gcc gtg tat tac tgt


 



gcg gca ga ! 1-58# 8 (SEQ ID NO: 41)


 



aga gtc acg att acc gcg gac gaa tcc acg agc aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 1-69# 9 (SEQ ID NO: 42)


 



aga gtc acg att acc gcg gac aaa tcc acg agc aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 1-e# 10 (SEQ ID NO: 43)


 



aga gtc acc ata acc gcg gac acg tct aca gac aca gcc tac atg


 



gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt


 



gcg aca ga ! 1-f# 11 (SEQ ID NO: 44)


 


!
VH2



agg ctc acc atc acc aag gac acc tcc aaa aac cag gtg gtc ctt


 



aca atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt


 



gca cac aga c! 2-05# 12 (SEQ ID NO: 45)


 



agg ctc acc atc tcc aag gac acc tcc aaa agc cag gtg gtc ctt


 



acc atg acc aac atg gac cct gtg gac aca gcc aca tat tac tgt


 



gca cgg ata c! 2-26# 13 (SEQ ID NO: 46)


 



agg ctc acc atc tcc aag gac acc tcc aaa aac cag gtg gtc ctt


 



aca atg acc aac atg gac cct gtg gac aca gcc acg tat tac tgt


 



gca cgg ata c! 2-70# 14 (SEQ ID NO: 47)


 


!
VH3



cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg


 



caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3-07# 15 (SEQ ID NO: 48)


 



cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg


 



caa atg aac agt ctg aga gct gag gac acg gcc ttg tat tac tgt


 



gca aaa gat a! 3-09# 16 (SEQ ID NO: 49)


 



cga ttc acc atc tcc agg gac aac gcc aag aac tca ctg tat ctg


 



caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 3-11# 17 (SEQ ID NO: 50)


 



cga ttc acc atc tcc aga gaa aat gcc aag aac tcc ttg tat ctt


 



caa atg aac agc ctg aga gcc ggg gac acg gct gtg tat tac tgt


 



gca aga ga ! 3-13# 18 (SEQ ID NO: 51)


 



aga ttc acc atc tca aga gat gat tca aaa aac acg ctg tat ctg


 



caa atg aac agc ctg aaa acc gag gac aca gcc gtg tat tac tgt


 



acc aca ga ! 3-15# 19 (SEQ ID NO: 52)


 



cga ttc acc atc tcc aga gac aac gcc aag aac tcc ctg tat ctg


 



caa atg aac agt ctg aga gcc gag gac acg gcc ttg tat cac tgt


 



gcg aga ga ! 3-20# 20 (SEQ ID NO: 53)


 



cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg


 



caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3-21# 21 (SEQ ID NO: 54)


 



cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg


 



caa atg aac agc ctg aga gcc gag gac acg gcc gta tat tac tgt


 



gcg aaa ga ! 3-23# 22 (SEQ ID NO: 55)


 



cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg


 



caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt


 



gcg aaa ga ! 3-30# 23 (SEQ ID NO: 56)


 



cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg


 



caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3303# 24 (SEQ ID NO: 57)


 



cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg


 



caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt


 



gcg aaa ga ! 3305# 25 (SEQ ID NO: 58)


 



cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg


 



caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3-33# 26 (SEQ ID NO: 59)


 



cga ttc acc atc tcc aga gac aac agc aaa aac tcc ctg tat ctg


 



caa atg aac agt ctg aga act gag gac acc gcc ttg tat tac tgt


 



gca aaa gat a! 3-43# 27 (SEQ ID NO: 60)


 



cga ttc acc atc tcc aga gac aat gcc aag aac tca ctg tat ctg


 



caa atg aac agc ctg aga gac gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3-48# 28 (SEQ ID NO: 61)


 



aga ttc acc atc tca aga gat ggt tcc aaa agc atc gcc tat ctg


 



caa atg aac agc ctg aaa acc gag gac aca gcc gtg tat tac tgt


 



act aga ga ! 3-49# 29 (SEQ ID NO: 62)


 



cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt


 



caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 3-53# 30 (SEQ ID NO: 63)


 



aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt


 



caa atg ggc agc ctg aga gct gag gac atg gct gtg tat tac tgt


 



gcg aga ga ! 3-64# 31 (SEQ ID NO: 64)


 



aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctt


 



caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt


 



gcg aga ga ! 3-66# 32 (SEQ ID NO: 65)


 



aga ttc acc atc tca aga gat gat tca aag aac tca ctg tat ctg


 



caa atg aac agc ctg aaa acc gag gac acg gcc gtg tat tac tgt


 



gct aga ga ! 3-72# 33 (SEQ ID NO: 66)


 



agg ttc acc atc tcc aga gat gat tca aag aac acg gcg tat ctg


 



caa atg aac agc ctg aaa acc gag gac acg gcc gtg tat tac tgt


 



act aga ca ! 3-73# 34 (SEQ ID NO: 67)


 



cga ttc acc atc tcc aga gac aac gcc aag aac acg ctg tat ctg


 



caa atg aac agt ctg aga gcc gag gac acg gct gtg tat tac tgt


 



gca aga ga ! 3-74# 35 (SEQ ID NO: 68)


 



aga ttc acc atc tcc aga gac aat tcc aag aac acg ctg cat ctt


 



caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt


 



aag aaa ga ! 3-d# 36 (SEQ ID NO: 69)


 


!
VH4



cga gtc acc ata tca gta gac aag tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4-04# 37 (SEQ ID NO: 70)


 



cga gtc acc atg tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gtg gac acg gcc gtg tat tac tgt


 



gcg aga aa ! 4-28# 38 (SEQ ID NO: 71)


 



cga gtt acc ata tca gta gac acg tct aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4301# 39 (SEQ ID NO: 72)


 



cga gtc acc ata tca gta gac agg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gcg gac acg gcc gtg tat tac tgt


 



gcc aga ga ! 4302# 40 (SEQ ID NO: 73)


 



cga gtt acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg act gcc gca gac acg gcc gtg tat tac tgt


 



gcc aga ga ! 4304# 41 (SEQ ID NO: 74)


 



cga gtt acc ata tca gta gac acg tct aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg act gcc gcg gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4-31# 42 (SEQ ID NO: 75)


 



cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gcg gac acg gct gtg tat tac tgt


 



gcg aga ga ! 4-34# 43 (SEQ ID NO: 76)


 



cga gtc acc ata tcc gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gca gac acg gct gtg tat tac tgt


 



gcg aga ca ! 4-39# 44 (SEQ ID NO: 77)


 



cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4-59# 45 (SEQ ID NO: 78)


 



cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gct gcg gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4-61# 46 (SEQ ID NO: 79)


 



cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg


 



aag ctg agc tct gtg acc gcc gca gac acg gcc gtg tat tac tgt


 



gcg aga ga ! 4-b# 47 (SEQ ID NO: 80)


 


!
VH5



cag gtc acc atc tca gcc gac aag tcc atc agc acc gcc tac ctg


 



cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt


 



gcg aga ca ! 5-51# 48 (SEQ ID NO: 81)


 



cac gtc acc atc tca gct gac aag tcc atc agc act gcc tac ctg


 



cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt


 



gcg aga ! 5-a# 49 (SEQ ID NO: 82)


 


!
VH6



cga ata acc atc aac cca gac aca tcc aag aac cag ttc tcc ctg


 



cag ctg aac tct gtg act ccc gag gac acg gct gtg tat tac tgt


 



gca aga ga ! 6-1# 50 (SEQ ID NO: 83)


 


!
VH7



cgg ttt gtc ttc tcc ttg gac acc tct gtc agc acg gca tat ctg


 



cag atc tgc agc cta aag gct gag gac act gcc gtg tat tac tgt


 



gcg aga ga ! 74.1# 51 (SEQ ID NO: 84)
















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







 1:  3  48:  3


There are 2 hits at base# 3








MaeIII 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








NlaIII 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








AluI 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


MwoI GCNNNNNnngc(SEQ ID NO: 85) 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








DdeI 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








BglII 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








Hpy188I 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 (SEQ ID NO: 86)
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








EaeI 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 Cggccg
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








HaeIII 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








HinfI Gantc
43







 2:  2   3:  2   4:  2   5:  2   6:  2   7:  2


 8:  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








MlyI GAGTCNNNNNn (SEQ ID NO: 87)
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








PleI 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








AciI 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








Bsp1286I 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








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








TseI Gcwgc
17







 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








MnlI gagg
34







 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


There are 1 hits at base# 52








AccI GTmkac
13 5-base 



recognition







 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








SacII CCGCgg
8 6-base 



recognition







 9: 14  10: 14  11: 14  37: 65  39: 65  40: 65


42: 65  43: 65


There are 5 hits at base# 65


There are 3 hits at base# 14








TfiI 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


26:  2  27:  2  28:  2  29:  2  30:  2  31:  2


32:  2  33:  2  33: 22  34: 22  35:  2  36:  2


There are 20 hits at base# 2








BsmAI Nnnnnngagac (SEQ ID NO: 88)
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








BpmI 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 (SEQ ID NO: 89)
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








BanII GRGCYc
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








Ecl136I 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








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 | XbaI  |).


! 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).


 


 





!








(SEQ ID NO: 91)


 





(SEQ ID NO: 90)








!






















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 (SEQ ID NOS 92-100, 


respectively, in order of appearance)


 !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


   8                                            5-a ctgcctacctgcagtggagcag


   9                                           3-49 tcgcctatctgcaaatgaacag


B: HpyCH4V REdaptors, Extenders, and Bridges


  B.1 REdaptors


! Cutting HC lower strand:










! TmKeller for 100 mM NaCl, zero formamide


SEQ


! Edapters for cleavage
Tmw
TmK
ID NO:





(ON_HCFR36-1)       5′-agttctcccTGCAgctgaactc-3′
68.0
64.5
92


(ON_HCFR36-lA)        5′-ttctcccTGCAgctgaactc-3′
62.0
62.5
residues





3-22 of 92


(ON_HCFR36-1B)       5′-ttctcccTGCAgctgaac-3′
56.0
59.9
residues





3-20 of 92


(ON_HCFR33-15)     5′-cgctgtatcTGCAaatgaacag-3′
64.0
60.8
96


(ON_HCFR33-15A)      5′-ctgtatcTGCAaatgaacag-3′
56.0
56.3
residues





3-22 of 96


(ON_HCFR33-15B)      5′-ctgtatcTGCAaatgaac-3′
50.0
53.1
residues





3-20 of 96


(ON_HCFR33-11)     5′-cactgtatcTGCAaatgaacag-3′
62.0
58.9
93


(ON_HCFR35-51)     5′-ccgcctaccTGCAgtggagcag-3′
74.0
70.1
95







!


  B.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned


!                    XbaI...                    (SEQ ID NO: 101)


!D323*  cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC


!       scab........ designed gene 3-23 gene................


!


!    HpyCH4V


!     .. ..            AflII...


!    Ttg caG atg aac agc TtA agG . . .


!    ........................... . . .


!


  B.3 Extender and Bridges


! Extender (bottom strand):


!                                                 (SEQ ID NO: 102)


(ON_HCHpyEx01) 5′-cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3′


! ON_HCHpyEx01 is the reverse complement of


! 5′-cgCttcacTaag tcT aga gac aaC tcT aag aaT acT ctC taC Ttg -3′


!


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


!                                            (SEQ ID NO: 103)


(ON_HCHpyBr016-1)   5′-cgCttcacTaag tcT aga gac aaC tcT aag-


                  aaT acT ctC taC Ttg CAgctgaac-3′ {3′-term C is blocked}


!


! 3-15 et al. + 3-11                         (SEQ ID NO: 104)


(ON_HCHpyBr023-15)  5′-cgCttcacTaag tcT aga gac aaC tcT aag-


                  aaT acT ctC taC Ttg CAaatgaac-3′ {3′-term C is blocked}


!


! 5-51                                       (SEQ ID NO: 105)


(ON_HCHpyBr045-51)  5′-cgCttcacTaag tcT aga gac aaC tcT aag-


                  aaT acT ctC taC Ttg CAgtggagc-3′ {3′-term C is blocked}


!


! PCR primer (top strand)


!


(ON_HCHpyPCR)       5′-cgCttcacTaag tcT aga gac-3′ (SEQ ID NO: 106)


!


C: BlpI Probes from human HC GLGs


   1              1-58, 1-03, 1-08, 1-69, 1-24, 1-45, 1-46, 1-f, 1-e acatggaGCTGAGCagcctgag


(SEQ ID NO: 107)


   2                                                            1-02 acatggaGCTGAGCaggctgag


(SEQ ID NO: 108)


   3                                                            1-18 acatggagctgaggagcctgag


(SEQ ID NO: 109)


   4                                                       5-51, 5-a acctgcagtggagcagcctgaa


(SEQ ID NO: 110)   


   5                                          3-15, 3-73, 3-49, 3-72 atctgcaaatgaacagcctgaa


(SEQ ID NO: 111)


   6            3303, 3-33, 3-07, 3-11, 3-30, 3-21, 3-23, 3305, 3-48 atctgcaaatgaacagcctgag


(SEQ ID NO: 112)


   7                                          3-20, 3-74, 3-09, 3-43 atctgcaaatgaacagtctgag


(SEQ ID NO: 113)


   8                                                            74.1 atctgcagatctgcagcctaaa


(SEQ ID NO: 114)


   9                                           3-66, 3-13, 3-53, 3-d atcttcaaatgaacagcctgag


(SEQ ID NO: 115)


  10                                                            3-64 atcttcaaatgggcagcctgag


(SEQ ID NO: 116)


  11 4301, 4-28, 4302, 4-04, 4304, 4-31, 4-34, 4-39, 4-59, 4-61, 4-b ccctgaaGCTGAGCtctgtgac


(SEQ ID NO: 117)


  12                                                             6-1 ccctgcagctgaactctgtgac


(SEQ ID NO: 118)


  13                                                      2-70, 2-05 tccttacaatgaccaacatgga


(SEQ ID NO: 119)


  14                                                            2-26 tccttaccatgaccaacatgga


(SEQ ID NO: 120)


D: BlpI REdaptors, Extenders, and Bridges


  D.1 REdaptors











Tmw
TmK



                        (SEQ ID NO: 121)





(BlpF3HC1-58)   5′-ac atg gaG CTG AGC agc ctg ag-3′
70
66.4



                              (SEQ ID NO: 122)





(BlpF3HC6-1)    5′-cc ctg aag ctg agc tct gtg ac-3′
70
66.4








! BlpF3HC6-1 matches 4-30.1, not 6-1.


  D.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned


!


BlpI


!                   XbaI...                                       . ...


...


!D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT ctC taC Ttg caG atg


aac


(SEQ ID NO: 123)!


!                   AflII...


!                 agC TTA AGG


  D.3 Extender and Bridges


! Bridges


 








(B1pF3Br1)






 


(B1pF3Br2)  ! 






 







! Extender


(BlpF3Ext) 5′-TcAgcTgcAAgTAcAAAgTATTTTTAcTgTTATcTcTAgAcTgAgTgAAgcg-3′ (SEQ


ID NO: 126)


! BlpF3Ext is the reverse complement of:


! 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3′


!


(BlpF3PCR) 5′-cgCttcacTcag tcT aga gaT aaC-3′ (SEQ ID NO: 127)


E: HpyCH4III Distinct GLG sequences surrounding site, bases 77-98


   1          102#1, 118#4, 146#7, 169#9, 1e#10, 311#17, 353#30, 404#37, 4301 ccgtgtattac


                                                                              tgtgcgagaga


(SEQ ID NO: 128)


   2          103#2, 307#15, 321#21, 3303#24, 333#26, 348#28, 364#31,  366#32 ctgtgtattac


                                                                              tgtgcgagaga


(SEQ ID NO: 129)


   3                                                                    108#3 ccgtgtattac


                                                                              tgtgcgagagg


(SEQ ID NO: 130)


   4                                                             124#5, 1f#11 ccgtgtattac


                                                                              tgtgcaacaga


(SEQ ID NO: 131)


   5                                                                    145#6 ccatgtattac


                                                                              tgtgcaagata


(SEQ ID NO: 132)


   6                                                                    158#8 ccgtgtattac


                                                                              tgtgcggcaga


(SEQ ID NO: 133)


   7                                                                   205#12 ccacatattac


                                                                              tgtgcacacag


(SEQ ID NO: 134)


   8                                                                   226#13 ccacatattac


                                                                              tgtgcacggat


(SEQ ID NO: 135)


   9                                                                   270#14 ccacgtattac


                                                                              tgtgcacggat


(SEQ ID NO: 136)


  10                                                           309#16, 343#27 ccttgtattac


                                                                              tgtgcaaaaga


(SEQ ID NO: 137)


  11                                                    313#18, 374#35, 61#50 ctgtgtattac


                                                                              tgtgcaagaga


(SEQ ID NO: 138)


  12                                                                   315#19 ccgtgtattac


                                                                              tgtaccacaga


(SEQ ID NO: 139)


  13                                                                   320#20 ccttgtatcac


                                                                              tgtgcgagaga


(SEQ ID NO: 140)


  14                                                                   323#22 ccgtatattac


                                                                              tgtgcgaaaga


(SEQ ID NO: 141)


  15                                                          330#23, 3305#25 ctgtgtattac


                                                                              tgtgcgaaaga


(SEQ ID NO: 142)


  16                                                                   349#29 ccgtgtattac


                                                                              tgtactagaga


(SEQ ID NO: 143)


  17                                                                   372#33 ccgtgtattac


                                                                              tgtgctagaga


(SEQ ID NO: 144)


  18                                                                   373#34 ccgtgtattac


                                                                              tgtactagaca


(SEQ ID NO: 145)


  19                                                                    3d#36 ctgtgtattac


                                                                              tgtaagaaaga


(SEQ ID NO: 146)


  20                                                                   428#38 ccgtgtattac


                                                                              tgtgcgagaaa


(SEQ ID NO: 147)


  21                                                         4302#40, 4304#41 ccgtgtattac


                                                                              tgtgccagaga


(SEQ ID NO: 148)


  22                                                                   439#44 ctgtgtattac


                                                                              tgtgcgagaca


(SEQ ID NO: 149)


  23                                                                   551#48 ccatgtattac


                                                                              tgtgcgagaca


(SEQ ID NO: 150)


  24                                                                    5a#49 ccatgtattac


                                                                              tgtgcgaga


(SEQ ID NO: 151)


F: HpyCH4III REdaptors, Extenders, and Bridges


  F.1 REdaptors


(SEQ ID NOS 152-159, respectively, in order of appearance)


! ONs for cleavage of HC(lower) in FR3(bases 77-97)


! For cleavage with HpyCH4III, Bst4CI, or TaaI


! cleavage is in lower chain before base 88.


!                      77 788 888 888 889 999 999 9









!                      78 901 234 567 890 123 456 7             
TmW TmK



(H43.77.97.1-02#1)  5′-cc gtg tat tAC TGT gcg aga g-3′ 
6462.6



(H43.77.97.1-03#2)  5′-ct 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 gta tat tac tgt gcg aaa g-3′
6058.7



(H43.77.97.330#23)  5′-ct gtg tat tac tgt gcg aaa g-3′
6058.7



(H43.77.97.439#44)  5′-ct gtg tat tac tgt gcg aga c-3′
6260.6



(H43.77.97.551#48)  5′-cc atg tat tac tgt gcg aga c-3′
6260.6



(H43.77.97.5a#49)   5′-cc atg tat tAC TGT gcg aga  -3′
5858.3








  F.2 Extender and Bridges


! XbaI and AflII sites in bridges are bunged


(H43.XABr1) 5′-ggtgtagtga-


 


 





 


(SEQ ID NO: 160)


(H43.XABr2) 5′-ggtgtagtga-


 


 





 


(SEQ ID NO: 161)


 


(H43.XAExt) 5′-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc TgcAAgTAgA-


               gAgTATTcTT AgAgTTgTcT cTAgATcAcT AcAcc-3′ (SEQ ID NO: 162)


!H43.XAExt is the reverse complement of


! 5′-ggtgtagtga-


 








 


(H43.XAPCR)  5′-ggtgtagtga |TCT|AGA|gac|aac-3′ (SEQ ID NO: 163)


! XbaI and AflII sites in bridges are bunged


(H43.ABr1) 5′-ggtgtagtga-


 


 





 


(SEQ ID NO: 164)


(H43.ABr2) 5′-ggtgtagtga-


 


 





 


(SEQ ID NO: 165)


(H43.AExt) 5′-ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3′


(SEQ ID NO: 166)


! (H43.AExt) is the reverse complement of 5′-ggtgtagtga-


 






















TABLE 5





Analysis of frequency of matching REdaptors in actual V genes







A: HpyCH4V in HC at bases 35-36














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

























Id
Ntot
0
1
2
3
4
5
6
7
8
9
10
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
  8
   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
  0
  2
  0
  1
   0
   0
   0
   0
   0
   4
   0
   3
7-4.1
cggcatatcTGCAgatctgcag


 


7
   7
  0
  2
  2
  0
   0
   2
   1
   0
   0
   0
   0
   4
3-73
cggcgtatcTGCAaatgaacag


 


8
  26
 10
  4
  1
  3
   1
   2
   1
   3
   1
   0
   0
  19
5-a
ctgcctaccTGCAgtggagcag


 


9
  21
  8
  2
  3
  1
   6
   1
   0
   0
   0
   0
   0
  20
3-49
tcgcctatcTGCAaatgaacag



1338
249
162
379
149
 103
 120
  71
  47
  13
  23
  12
1052

(SEQ ID NOS 169-177,




249
411
790
939

1162

1280

1316



repectively in order








1042

1233

1293

1338


of appearance)












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










(SEQ ID NOS 169-177, respectively in order of appearance)


 


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: BlpI in HC




















Id
Ntot
0
1
2
3
4
5
6
7
8
Ncut
Name






 1
133
 73
16
11
13
 6
 9
1
4
0
119
1-58
acatggaGCTGAGCagcctgag


 


 2
 14
 11
 1
 0
 0
 0
 0
1
0
1
 12
1-02
acatggagctgagcaggctgag


 


 3
 34
 17
 8
 2
 6
 1
 0
0
0
0
  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
atctgcaatgaacagcctgag


 


 7
 82
 25
16
25
12
 1
 3
0
0
0
  0
3-20
atctgcaaatgaacagtctgag


 


 8
  3
  0
 2
 0
 1
 0
 0
0
0
0
  0
74.1
atctgcagatctgcagcctaaa


 


 9
 23
 18
 2
 2
 1
 0
 0
0
0
0
  0
3-66
atcttcaaatgaacagcctgag


 


10
  2
  1
 0
 1
 0
 0
 0
0
0
0
  0
3-64
atcttcaaatgggcagcctgag


 


11
486
249
78
81
38
21
10
4
4
1
467
4301
ccctgaagctgagctctgtgac


 


12
 16
  6
 3
 1
 0
 1
 1
3
1
0
  1
6-1
ccctgcagctgaactctgtgac


 


13
 28
 15
 8
 2
 2
 1
 0
0
0
0
  0
2-70
tccttacaatgaccaacatgga


 


14
  2
  0
 2
 0
 0
 0
 0
0
0
0
  0
2-26
tccttaccatgaccaacatgga













601

(SEQ ID NOS 178-191),















respectively in order















of appearance)












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










(SEQ ID NOS 178-191, respectively in order of appearance)


 


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: HpyCH4III,Bst4CI, or TaaI in HC


In scoring whether the RE site of interest is present,


only ONs that have 4 or fewer mismatches are counted.


Number of sequences.......... 1617




















Id
Ntot
0
1
2
3
4
5
6
7
8
Ncut

acngt





 1
244
 78
 92
  43
  18
  10
   1
   2
   0
   0
241
102#1,1
ccgtgtattACTGTgcgagaga


 


 2
457
 69
150
 115
  66
  34
  11
   8
   3
   1
434
103#2,3
ctgtgtattactgtgcgagaga


 


 3
173
 52
 45
  36
  22
  14
   3
   0
   0
   1
169
108#3
ccgtgtattactgtgcgagagg


 


 4
 16
  0
  3
   2
   2
   1
   6
   0
   1
   1
  8
124#5,1
ccgtgtattactgtgcaacaga


 


 5
  4
  0
  0
   1
   0
   1
   1
   0
   1
   0
  2
145#6
ccatgtattactgtgcaagata


 


 6
 15
  1
  0
   1
   0
   6
   4
   1
   1
   1
  8
158#8
ccgtgtattactgtgcggcaga


 


 7
 23
  4
  8
   5
   2
   2
   1
   1
   0
   0
 21
205#12
ccacatattactgtgcacacag


 


 8
  9
  1
  1
   1
   0
   3
   2
   1
   0
   0
  6
226#13
ccacatattactgtgcacggat


 


 9
  7
  1
  3
   1
   1
   0
   0
   1
   0
   0
  6
270#14
ccacgtattactgtgcacggat


 


10
 23
  7
  3
   5
   5
   2
   1
   0
   0
   0
 22
309#16,
ccttgtattactgtgcaaaaga


 


11
 35
  5
 10
   7
   6
   3
   3
   0
   1
   0
 31
313#18,
ctgtgtattactgtgcaagaga


 


12
 18
  2
  3
   2
   2
   6
   1
   0
   2
   0
 15
315#19
ccgtgtattactgtaccacaga


 


13
  3
  1
  2
   0
   0
   0
   0
   0
   0
   0
  3
320#20
ccttgtatcactgtgcgagaga


 


14
117
 29
 23
  28
  22
   8
   4
   2
   1
   0
110
323#22
ccgtatattactgtgcgaaaga


 


15
 75
 21
 25
  13
   9
   1
   4
   2
   0
   0
 69
330#23,
ctgtgtattactgtgcgaaaga


 


16
 14
  2
  2
   2
   3
   0
   3
   1
   1
   0
  9
349#29
ccgtgtattactgtactagaga


 


17
  2
  0
  0
   1
   0
   0
   1
   0
   0
   0
  1
372#33
ccgtgtattactgtgctagaga


 


18
  1
  0
  0
   1
   0
   0
   0
   0
   0
   0
  1
373#34
ccgtgtattactgtactagaca


 


19
  2
  0
  0
   0
   0
   0
   0
   0
   0
   2
  0
3d#36
ctgtgtattactgtaagaaaga


 


20
 34
  4
  9
   9
   4
   5
   3
   0
   0
   0
 31
428#38
ccgtgtattactgtgcgagaaa


 


21
 17
  5
  4
   2
   2
   3
   1
   0
   0
   0
 16
4302#40
ccgtgtattactgtgccagaga


 


22
 75
 15
 17
  24
   7
  10
   1
   1
   0
   0
 73
439#44
ctgtgtattactgtgcgagaca


 


23
 40
 14
 15
   4
   5
   1
   0
   1
   0
   0
 39
551#48
ccatgtattactgtgcgagaca


 


24
213
 26
 56
  60
  42
  20
   7
   2
   0
   0
204
5a#49
ccatgtattactgtgcgagaAA


 


















Group
337
471
 363
 218
 130
  58
  23
  11
   6

(SEQ ID NOS 192-215, respectively


Cumulative
337
808
1171
1389
1519
1577
1600
1611
1617

in order of appearance)













acngt
acngt





102#1,1
ccgtgtattACTGTgcgagaga
ccgtgtattactgtgcgagaga


 


103#2,3
ctgtgtattactgtgcgagaga
.t....................


 


108#3
ccgtgtattactgtgcgagagg
.....................g


 


124#5,1
ccgtgtattactgtgcaacaga
................a.c...


 


145#6
ccatgtattactgtgcaagata
..a.............a...t.


 


158#8
ccgtgtattactgtgcggcaga
.................gc...


 


205#12
ccacatattactgtgcacacag
..aca...........acacag


 


226#13
ccacatattactgtgcacggat
..aca...........ac.gat


 


270#14
ccacgtattactgtgcacggat
..ac............ac.gat


 


309#16,
ccttgtattactgtgcaaaaga
..t.............a.a...


 


313#18,
ctgtgtattactgtgcaagaga
.t..............a.....


 


315#19
ccgtgtattactgtaccacaga
..............a.c.c...


 


320#20
ccttgtatcactgtgcgagaga
..t.....c.............


 


323#22
ccgtatattactgtgcgaaaga
....a.............a...


 


330#23,
ctgtgtattactgtgcgaaaga
.t................a...


 


349#29
ccgtgtattactgtactagaga
..............a.t.....


 


372#33
ccgtgtattactgtgctagaga
................t.....


 


373#34
ccgtgtattactgtactagaca
..............a.t...c.


 


3d#36
ctgtgtattactgtaagaaaga
.t............aa..a...


 


428#38
ccgtgtattactgtgcgagaaa
....................a.


 


4302#40
ccgtgtattactgtgccagaga
................c.....


 


439#44
ctgtgtattactgtgcgagaca
.t..................c.


 


551#48
ccatgtattactgtgcgagaca
..a.................c.


 


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










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
0
1
2
3
4
5
6
7
8+

























1
301
78
101
 54
32
16
 9
10
 1
0
281
102#1
ccgtgtattactgtgcgagaga
(SEQ ID NO: 267)


 


2
493
69
155
125
73
37
14
11
 3
6
459
103#2
ctgtgtattactgtgcgagaga
(SEQ ID NO: 268)


 


3
189
52
 45
 38
23
18
 5
 4
 1
3
176
108#3
ccgtgtattactgtgcgagagg
(SEQ ID NO: 269)


 


4
127
29
 23
 28
24
10
 6
 5
 2
0
114
323#22
ccgtatattactgtgcgaaaga
(SEQ ID NO: 270)


 


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
(SEQ ID NO: 272)


 


7
 43
14
 15
  5
 5
 3
 0
 1
 0
0
 42
551#48
ccatgtattactgtgcgagaca
(SEQ ID NO: 273)


 


8
307
26
 63
 72
51
38
24
14
13
6
250
5a#49
ccatgtattactgtgcgaga
(residues 1-20  
















of SEQ
















ID NO: 274)













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










(SEQ ID NOS 267-274, respectively in order of appearance)


 


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 (SEQ ID NO: 216)


 


1-03
cag gtC cag ctT gtg cag tct ggg gct gag gtg aag aag cct ggg gcc tca gtg aag



gtT tcc tgc aag gct tct gga tac acc ttc acT (SEQ ID NO: 217)


 


1-08
cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg gcc tca gtg aag



gtc tcc tgc aag gct tct gga tac acc ttc acc (SEQ ID NO: 218)


 


1-18
cag gtT cag ctg gtg cag tct ggA gct gag gtg aag aag cct ggg gcc tca gtg aag



gtc tcc tgc aag get tct ggT tac acc ttT acc (SEQ ID NO: 219)


 


1-24
cag gtC cag ctg gtA cag tct ggg get gag gtg aag aag cct ggg gcc tca gtg aag



gtc tcc tgc aag gTt tcC gga tac acc Ctc acT (SEQ ID NO: 220)


 


1-45
cag Atg cag ctg gtg cag tct ggg get gag gtg aag aag Act ggg Tcc tca gtg aag



gtT tcc tgc aag get tcC gga tac acc ttc acc (SEQ ID NO: 221)


 


1-46
cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg gcc tca gtg aag



gtT tcc tgc aag gcA tct gga tac acc ttc acc (SEQ ID NO: 222)


 


1-58
caA Atg cag ctg gtg cag tct ggg Cct gag gtg aag aag cct ggg Acc tca gtg aag



gtc tcc tgc aag get tct gga tTc acc ttT acT (SEQ ID NO: 223)


 


1-69
cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg Tcc tcG gtg aag



gtc tcc tgc aag get tct gga GGc acc ttc aGc (SEQ ID NO: 224)


 


1-e
cag gtg cag ctg gtg cag tct ggg get gag gtg aag aag cct ggg Tcc tcG gtg aag



gtc tcc tgc aag get tct gga GGc acc ttc aGc (SEQ ID NO: 225)


 


1-f
Gag gtC cag ctg gtA cag tct ggg get gag gtg aag aag cct ggg gcT Aca gtg aaA



Atc tcc tgc aag gTt tct gga tac acc ttc acc (SEQ ID NO: 226)


 







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 (SEQ ID NO: 227)


 


2-26 
cag Gtc acc ttg aag gag tct ggt cct GTg ctg gtg aaa ccc aca Gag acc ctc acg



ctg acc tgc acc Gtc tct ggg ttc tca ctc agc (SEQ ID NO: 228)


 


2-70 
cag Gtc acc ttg aag gag tct ggt cct Gcg ctg gtg aaa ccc aca cag acc ctc acA



ctg acc tgc acc ttc tct ggg ttc tca ctc agc (SEQ ID NO: 229)


 







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 (SEQ ID NO: 230)


 


3-09
gaA gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggC Agg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttt Gat (SEQ ID NO: 231)


 


3-11
Cag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc Aag cct ggA ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 232)


 


3-13
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 233)


 


3-15
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA Aag cct ggg ggg tcc ctT aga



ctc tcc tgt gca gcc tct gga ttc acT ttC agt (SEQ ID NO: 234)


 


3-20
gag gtg cag ctg gtg gag tct ggg gga ggT Gtg gtA cGg cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttt Gat (SEQ ID NO: 235)


 


3-21
gag gtg cag ctg gtg gag tct ggg gga ggc Ctg gtc Aag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 236)


 


3-23
gag gtg cag ctg Ttg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttt agC (SEQ ID NO: 237)


 


3-30
Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 238)


 


3-30.3 
Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 239)


 


3-30.5 
Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 240)


 


3-33
Cag gtg cag ctg gtg gag tct ggg gga ggc Gtg gtc cag cct ggg Agg tcc ctg aga



ctc tcc tgt gca gcG tct gga ttc acc ttC agt (SEQ ID NO: 241)


 


3-43
gaA gtg cag ctg gtg gag tct ggg gga gTe Gtg gtA cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttt Gat (SEQ ID NO: 242)


 


3-48
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 243)


 


3-49
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtA cag ccA ggg Cgg tcc ctg aga



ctc tcc tgt Aca gcT tct gga ttc acc ttt Ggt (SEQ ID NO: 244)


 


3-53
gag gtg cag ctg gtg gag Act ggA gga ggc ttg Atc cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct ggG ttc acc GtC agt (SEQ ID NO: 245)


 


3-64
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 ttC agt (SEQ ID NO: 246)


 


3-66
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 GtC agt (SEQ ID NO: 247)


 


3-72
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggA ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 248)


 


3-73
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gtc cag cct ggg ggg tcc ctg aAa



ctc tcc tgt gca gcc tct ggG ttc acc ttC agt (SEQ ID NO: 249)


 


3-74
gag gtg cag ctg gtg gag tcC ggg gga ggc ttA gtT cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc ttC agt (SEQ ID NO: 250)


 


3-d
gag gtg cag ctg gtg gag tct Cgg gga gTc ttg gtA cag cct ggg ggg tcc ctg aga



ctc tcc tgt gca gcc tct gga ttc acc GtC agt (SEQ ID NO: 251)


 







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 (SEQ ID NO: 252)


 


4-28
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAC acc ctg tcc



ctc acc tgc get gtc tct ggt TAc tcc atc agc (SEQ ID NO: 253)


 


4-30.1
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc atc agc (SEQ ID NO: 254)


 


4-30.2
cag Ctg cag ctg cag gag tcC ggc Tca gga ctg gtg aag cct tcA CAg acc ctg tcc



ctc acc tgc gct gtc tct ggt ggc tcc atc agc (SEQ ID NO: 255)


 


4-30.4
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc atc agc (SEQ ID NO: 256)


 


4-31
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcA CAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc atc agc (SEQ ID NO: 257)


 


4-34
cag gtg cag ctA cag Cag tGg ggc Gca gga ctg Ttg aag cct tcg gAg acc ctg tcc



ctc acc tgc gct gtc tAt ggt ggG tcc Ttc agT (SEQ ID NO: 258)


 


4-39
cag Ctg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc atc agc (SEQ ID NO: 259)


 


4-59
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc atc agT (SEQ ID NO: 260)


 


4-61
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc



ctc acc tgc Act gtc tct ggt ggc tcc Gtc agc (SEQ ID NO: 261)


 


4-b
cag gtg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gAg acc ctg tcc



ctc acc tgc gct gtc tct ggt TAC tcc atc agc (SEQ ID NO: 262)


 







VH5








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



ATC TCC TGT AAG GGT TCT GGA TAC AGC TTT ACC (SEQ ID NO: 263)


 


5-a
gaA gtg cag ctg gtg cag tct gga gca gag gtg aaa aag ccc ggg gag tct ctg aGg



atc tcc tgt aag ggt tct gga tac agc ttt acc (SEQ ID NO: 264)


 







VH6








6-1
CAG GTA CAG CTG CAG CAG TCA GGT CCA GGA CTG GTG AAG CCC TCG CAG ACC CTC TCA



CTC ACC TGT GCC ATC TCC GGG GAC AGT GTC TCT (SEQ ID NO: 265)


 







VH7








7-4.1
CAG GTG CAG CTG GTG CAA TCT GGG TCT GAG TTG AAG AAG CCT GGG GCC TCA GTG AAG



GTT TCC TGC AAG GCT TCT GGA TAC ACC TTC ACT (SEQ ID NO: 266)
















TABLE 7





RERS sites in Human HC GLG FR1s 


where there are at least 20 GLGs cut
















BsgI GTGCAG
71 



(cuts 16/14 bases to right)







 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








BbvI 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 sit s


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








TseI Gcwgc
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 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








MspA1I 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








AluI 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# 8


There are 2 hits at base# 2








DdeI 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($M.HpaII) 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








NlaIV 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 & 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


(SEQ ID NO: 13)








BsmAI 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


(SEQ ID NO: 14)








-′′- 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








AvaII 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 & 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








HinfI 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








TfiI 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








MlyI 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








PleI 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


















!
1   2   3   4   5   6   7   8   9   10  11  12





GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
O12
(SEQ ID NO: 275)


 


!
13  14  15  16  17  18  19  20  21  22  23





GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
O2
(SEQ ID NO: 276)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
O18
(SEQ ID NO: 277)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
O8
(SEQ ID NO: 278)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
A20
(SEQ ID NO: 279)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
A30
(SEQ ID NO: 280)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



AAC ATC CAG ATG ACC CAG TCT CCA TCT GCC ATG TCT
L14
(SEQ ID NO: 281)


 



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




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT
L1
(SEQ ID NO: 282)


 



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




 



GAC ATC CAG ATG ACC CAG TCT CCA TCC TCA CTG TCT
L15
(SEQ ID NO: 283)


 



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




 



GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT
L4
(SEQ ID NO: 284)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GCC ATC CAG TTG ACC CAG TCT CCA TCC TCC CTG TCT
L18
(SEQ ID NO: 285)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT
L5
(SEQ ID NO: 286)


 



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




 



GAC ATC CAG ATG ACC CAG TCT CCA TCT TCT GTG TCT
L19
(SEQ ID NO: 287)


 



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




 



GAC ATC CAG TTG ACC CAG TCT CCA TCC TTC CTG TCT
L8
(SEQ ID NO: 288)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GCC ATC CGG ATG ACC CAG TCT CCA TTC TCC CTG TCT
L23
(SEQ ID NO: 289)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GCC ATC CGG ATG ACC CAG TCT CCA TCC TCA TTC TCT
L9
(SEQ ID NO: 290)


 



GCA TCT ACA GGA GAC AGA GTC ACC ATC ACT TGT  !




 



GTC ATC TGG ATG ACC CAG TCT CCA TCC TTA CTC TCT
L24
(SEQ ID NO: 291)


 



GCA TCT ACA GGA GAC AGA GTC ACC ATC AGT TGT  !




 



GCC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT
L11
(SEQ ID NO: 292)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAC ATC CAG ATG ACC CAG TCT CCT TCC ACC CTG TCT
L12
(SEQ ID NO: 293)


 



GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC  !




 



GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC
O11
(SEQ ID NO: 294)


 



GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC CTG CCC
O1
(SEQ ID NO: 295)


 



GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !




 



GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
A17
(SEQ ID NO: 296)


 



GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC  !




 



GAT GTT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
A1
(SEQ ID NO: 297)


 



GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC
A18
(SEQ ID NO: 298)


 



GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACC CAG ACT CCA CTC TCT CTG TCC
A2
(SEQ ID NO: 299)


 



GTC ACC CCT GGA CAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
A19
(SEQ ID NO: 300)


 



GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACT CAG TCT CCA CTC TCC CTG CCC
A3
(SEQ ID NO: 301)


 



GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC  !




 



GAT ATT GTG ATG ACC CAG ACT CCA CTC TCC TCA CCT
A23
(SEQ ID NO: 302)


 



GTC ACC CTT GGA CAG CCG GCC TCC ATC TCC TGC  !




 



GAA ATT GTG TTG ACG CAG TCT CCA GGC ACC CTG TCT
A27
(SEQ ID NO: 303)


 



TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATT GTG TTG ACG CAG TCT CCA GCC ACC CTG TCT
A11
(SEQ ID NO: 304)


 



TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT
L2
(SEQ ID NO: 305)


 



GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT
L16
(SEQ ID NO: 306)


 



GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT
L6
(SEQ ID NO: 307)


 



TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATT GTG TTG ACA CAG TCT CCA GCC ACC CTG TCT
L20
(SEQ ID NO: 308)


 



TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAA ATT GTA ATG ACA CAG TCT CCA GCC ACC CTG TCT
L25
(SEQ ID NO: 309)


 



TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC  !




 



GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT
B3
(SEQ ID NO: 310)


 



GTG TCT CTG GGC GAG AGG GCC ACC ATC AAC TGC  !




 



GAA ACG ACA CTC ACG CAG TCT CCA GCA TTC ATG TCA
B2
(SEQ ID NO: 311)


 



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




 



GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT
A26
(SEQ ID NO: 312)


 



GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC  !




 



GAA ATT GTG CTG ACT CAG TCT CCA GAC TTT CAG TCT
A10
(SEQ ID NO: 313)


 



GTG ACT CCA AAG GAG AAA GTC ACC ATC ACC TGC  !




 



GAT GTT GTG ATG ACA CAG TCT CCA GCT TTC CTC TCT
A14
(SEQ ID NO: 314)


 



GTG ACT CCA GGG GAG AAA GTC ACC ATC ACC TGC  !
















TABLE 9





RERS sites found in Human Kappa FR1 GLGs


























FokI




HpyCH




MslI
−−> <−− −−>
PflFI
BsrI
BsmAI
MnlI
4V





VKI










O12
  1-69
  3
  3  23
 12  49
 15
 18  47
 26
 36


O2
 101-169
 103
 103  123
 112  149
 115
 118  147
 126
 136


O18
 201-269
 203
 203  223
 212  249
 215
 218  247
 226
 236


O8
 301-369
 303
 303  323
 312  349
 315
 318  347
 326
 336


A20
 401-469
 403
 403  423
 412  449
 415
 418  447
 426
 436


A30
 501-569
 503
 503  523
 512  549
 515
 518  547
 526
 536


L14
 601-669
 603
 603
 612  649
 615
 618  647
—
 636


L1
 701-769
 703
 703  723
 712  749
 715
 718  747
 726
 736


L15
 801-869
 803
 803  823
 812  849
 815
 818  847
 826
 836


L4
 901-969
—
 903  923
 912  949
 906  915
 918  947
 926
 936


L18
1001-1069
—
1003
1012 1049
1006 1015
1018 1047
1026
1036


L5
1101-1169
1103
— 
1112 1149
1115
1118 1147
—
1136


L19
1201-1269
1203
1203
1212 1249
1215
1218 1247
—
1236


L8
1301-1369
—
1303 1323
1312 1349
1306 1315
1318 1347
—
1336


L23
1401-1469
1403
1403 1408
1412 1449
1415
1418 1447
—
1436


L9
1501-1569
1503
1503 1508 1523
1512 1549
1515
1518 1547
1526
1536


L24
1601-1669
1603
1608 1623
1612 1649
1615
1618 1647
—
1636


L11
1701-1769
1703
1703 1723
1712 1749
1715
1718 1747
1726
1736


L12
1801-1869
1803
1803
1812 1849
1815
1818 1847
—
1836


VKII










O11
1901-1969
—
—
—
—
—
1956
—


O1
2001-2069
—
—
—
—

2056
—


A17
2101-2169
—
—
2112
—
2118
2156
—


A1
2201-2269
—
—
2212
—
2218
2256
—


A18
2301-2369
—
—
—
—
—
2356
—


A2
2401-2469
—
—
—
—
—
2456
—


A19
2501-2569
—
—
2512
—
2518
2556
—


A3
2601-2669
—
—
2612
—
2618
2656
—


A23
2701-2769
—
—
—
—
—
2729 2756
—


VKIII










A27
2801-2869
—
—
2812
—
2818 2839
2860
—


A11
2901-2969
—
—
2912
—
2918 2939
2960
—


L2
3001-3069
—
—
3012
—
3018 3039
3060
—


L16
3101-3169
—
—
3112
—
3118 3139
3160
—


L6
3201-3269
—
—
3212
—
3218 3239
3260
—


L20
3301-3369
—
—
3312
—
3318 3339
3360
—


L25
3401-3469
—
—
3412
—
3418 3439
3460
—


VKIV










B3
3501-3569
3503
—
3512
3515
3518 3539
3551<
—


VKV










B2
3601-3669
—
—
3649
—
3618 3647

—


VKVI










A26
3701-3769
—
—
3712
—
3718

—


A10
3801-3869
—
—
3812
—
3818

—


A14
3901-3969
—
—
3912
—
3918
3930>
—











MaeIII
HphI
HpaII







MlyI
Tsp45I same
xx38 xx56
MspI




SfaNI
SfcI
HinfI
−−> −−> <−−
sites
xx62
xx06 xx52





VKI










O12
  1-69
 37
 41
 53
 53
 55
 56
—


O2
 101-169
 137
 141
 153
 153
 155
 156
—


O18
 201-269
 237
 241
 253
 253
 255
 256
—


O8
 301-369
 337
 341
 353
 353
 355
 356
—


A20
 401-469
 437
 441
 453
 453
 455
 456
—


A30
 501-569
 537
 541
 553
 553
 555
 556
—


L14
 601-669
 637
 641
 653
 653
 655
 656
—


L1
 701-769
 737
 741
 753
 753
 755
 756
—


L15
 801-869
 837
 841
 853
 853
 855
 856
—


L4
 901-969
 937
 941
 953
 953
 955
 956
—


L18 
1001-1069
1037
1041
1053
1053
1055
1056
—


L5
1101-1169
1137
1141
1153
1153
1155
1156
—


L19 
1201-1269
1237
1241
1253
1253
1255
1256
—


L8
1301-1369
1337
1341
1353
1353
1355
1356
—


L23 
1401-1469
1437
1441
1453
1453
1455
1456
1406


L9
1501-1569
1537
1541
1553
1553
1555
1556
1506


L24 
1601-1669
1637
1641
1653
1653
1655
1656



L11 
1701-1769
1737
1741
1753
1753
1755
1756



L12 
1801-1869
1837
1841
1853
1853
1855
1856



VKII










O11 
1901-1969
—
—
1918
1918
1937
1938
1952


O1
2001-2069
—
—
2018
2018
2037
2038
2052


A17
2101-2169
—
—
2112
2112
2137
2138
2152


A1
2201-2169
—
—
2212
2212
2237
2238
2252


A18 
2301-2369
—
—
2318
2318
2337
2338
2352


A2 
2401-2469
—
—
2418
2418
2437
2438
2452


A19 
2501-2569
—
—
2512
2512
2537
2538
2552


A3
2601-2669
—
—
2612
2612
2637
2638
2652


A23 
2701-2769
—
—
2718
2718
2737
2731* 2738*
—


VKIII










A27 
2801-2869
—
—
—
—


—


A11
2901-2969
—
—
—
—


—


L2 
3001-3069
—
—
—
—


—


L16 
3101-3169
—
—
—
—


—


L6 
3201-3269
—
—
—
—


—


L20 
3301-3369
—
—
—
—


—


L25 
3401-3469
—
—
—
—


—


VKIV










B3 
3501-3569
—
—
3525
3525


—









VKV

















B2 
3601-3669
—
—
3639
3639


—














VKVI






















A26
3701-3769
—
—
3712 3739
3712 3739
3737 3755
3756 3762
—


A10 
3801-3869
—
—
3812 3839
3812 3839
3837 3855
3856 3862
—


A14
3901-3969
—
—
3939
3939
3937 3955
3956 3962
—






















BsrFI








BpmI
Cac8I






BsaJI
BssKI (NstNI)
xx20 xx41 xx44 
NaeI






xx29 xx42 xx43
xx22 xx30 xx43
−−> −−> <−−
NgoMIV
HaeIII
Tsp509I





VKI









O12
  1-69
—
—
—
—
—
—


O2
 101-169
—
—
—
—
—
—


O18 
 201-269
—
—
—
—
—
—


O8
 301-369
—
—
—
—
—
—


A20 
 401-469
—
—
—
—
—
—


A30 
 501-569
—
—
—
—
—
—


L14 
 601-669
—
—
—
—
—
—


L1
 701-769
—
—
—
—
—
—


L15 
 801-869
—
—
—
—
—
—


L4
 901-969
—
—
—
—
—
—


L18 
1001-1069
—
—
—
—
—
—


L5 
1101-1169
—
—
—
—
—
—


L19 
1201-1269
—
—
—
—
—
—


L8 
1301-1369
—
—
—
—
—
—


L23 
1401-1469
—
—
—
—
—
—


L9
1501-1569
—
—
—
—
—
—


L24 
1601-1669
—
—
—
—
—
—


L11 
1701-1769
—
—
—
—
—
—


L12 
1801-1869
—
—
—
—
—
—


VKII









O11 
1901-1969
1942
1943
1944
1951
1954
—


O1
2001-2069
2042
2043
2044
2051
2054
—


A17 
2101-2169
2142
—
—
2151
2154
—


A1
2201-2269
2242
—
—
2251
2254
—


A18 
2301-2369
2342
2343
—
2351
2354
—


A2 
2401-2469
2442
2443
—
2451
2454
—


A19 
2501-2569
2542
2543
2544
2551
2554
—


A3
2601-2669
2642
2643
2644
2651
2654
—


A23 
2701-2769
2742
—
—
2751
2754
—


VKIII









A27 
2801-2869
2843
2822 2843
2820 2841
—
—
2803


A11
2901-2969
2943
2943
2920 2941
—
—
2903


L2 
3001-3069
3043
3043
3041
—
—
—


L16 
3101-3169
3143
3143
3120 3141
—
—
—


L6 
3201-3269
3243
3243
3220 3241
—
—
3203


L20 
3301-3369
3343
3343
3320 3341
—
—
3303


L25
3401-3469
3443
3443
3420 3441
—
—
3403


VKIV









B3 
3501-3569
3529
3530
3520
—
3554



VKV









B2 
3601-3669

3643
3620 3641
—
—



VKVI









A26 
3701-3769

—
3720
—
—
3703


A10 
3801-3869

—
3820
—
—
3803


A14 
3901-3969
3943
3943
3920 3941
—
—
—

















TABLE 10





Lambda FR1 GLG sequences



















! VL1
CAG TCT GTG CTG ACT CAG CCA CCC TCG GTG TCT GAA
1a
(SEQ ID NO: 315)



GCC CCC AGG CAG AGG GTC ACC ATC TCC TGT !




 



cag tct gtg ctg acG cag ccG ccc tcA gtg tct gGG
1e
(SEQ ID NO: 316)



gcc ccA Ggg cag agg gtc acc atc tcc tgC !




 



cag tct gtg ctg act cag cca ccc tcA gCg tct gGG
1c
(SEQ ID NO: 317)



Acc ccc Ggg cag agg gtc acc atc tcT tgt !




 



cag tct gtg ctg act cag cca ccc tcA gCg tct gGG
1g
(SEQ ID NO: 318)



Acc ccc Ggg cag agg gtc acc atc tcT tgt !




 



cag tct gtg Ttg acG cag ccG ccc tcA gtg tct gCG
1b
(SEQ ID NO: 319)



gcc ccA GgA cag aAg gtc acc atc tcc tgC !




 


! VL2
CAG TCT GCC CTG ACT CAG CCT CCC TCC GCG TCC GGG
2c
(SEQ ID NO: 320)



TCT CCT GGA CAG TCA GTC ACC ATC TCC TGC !




 



cag tct gcc ctg act cag cct cGc tcA gTg tcc ggg
2e
(SEQ ID NO: 321)



tct cct gga cag tca gtc acc atc tcc tgc !




 



cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg
2a2
(SEQ ID NO: 322)



tct cct gga cag tcG Atc acc atc tcc tgc !




 



cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg
2d
(SEQ ID NO: 323)



tct cct gga cag tca gtc acc atc tcc tgc !




 



cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg
2b2
(SEQ ID NO: 324)



tct cct gga cag tcG Atc acc atc tcc tgc !




 


! VL3
TCC TAT GAG CTG ACT CAG CCA CCC TCA GTG TCC GTG
3r
(SEQ ID NO: 325)



TCC CCA GGA CAG ACA GCC AGC ATC ACC TGC !




 



tcc tat gag ctg act cag cca cTc tca gtg tcA gtg
3j
(SEQ ID NO: 326)



Gcc cTG gga cag acG gcc agG atT acc tgT !




 



tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg
3p
(SEQ ID NO: 327)



tcc cca gga caA acG gcc agG atc acc tgc !




 



tcc tat gag ctg acA cag cca ccc tcG gtg tcA gtg
3a
(SEQ ID NO: 328)



tcc cTa gga cag aTG gcc agG atc acc tgc !




 



tcT tCt gag ctg act cag GAC ccT GcT gtg tcT gtg
3l
(SEQ ID NO: 329)



Gcc TTG gga cag aca gTc agG atc acA tgc !




 



tcc tat gTg ctg act cag cca ccc tca gtg tcA gtg
3h
(SEQ ID NO: 330)



Gcc cca gga Aag acG gcc agG atT acc tgT !




 



tcc tat gag ctg acA cag cTa ccc tcG gtg tcA gtg
3e
(SEQ ID NO: 331)



tcc cca gga cag aca gcc agG atc acc tgc !




 



tcc tat gag ctg aTG cag cca ccc tcG gtg tcA gtg
3m
(SEQ ID NO: 332)



tcc cca gga cag acG gcc agG atc acc tgc !




 



tcc tat gag ctg acA cag cca Tcc tca gtg tcA gtg
V2-19
(SEQ ID NO: 333)



tcT ccG gga cag aca gcc agG atc acc tgc !




 


! VL4
CTG CCT GTG CTG ACT CAG CCC CCG TCT GCA TCT GCC
4c
(SEQ ID NO: 334)



TTG CTG GGA GCC TCG ATC AAG CTC ACC TGC !




 



cAg cct gtg ctg act caA TcA TcC tct gcC tct gcT
4a
(SEQ ID NO: 335)



tCC ctg gga Tcc tcg Gtc aag ctc acc tgc !




 



cAg cTt gtg ctg act caA TcG ccC tct gcC tct gcc
4b
(SEQ ID NO: 336)



tCC ctg gga gcc tcg Gtc aag ctc acc tgc !




 


! VL5
CAG CCT GTG CTG ACT CAG CCA CCT TCC TCC TCC GCA
5e
(SEQ ID NO: 337)



TCT CCT GGA GAA TCC GCC AGA CTC ACC TGC !




 



cag Gct gtg ctg act cag ccG Gct tcc CTc tcT gca
5c
(SEQ ID NO: 338)



tct cct gga gCa tcA gcc agT ctc acc tgc !




 



cag cct gtg ctg act cag cca Tct tcc CAT tcT gca
5b
(SEQ ID NO: 339)



tct Tct gga gCa tcA gTc aga ctc acc tgc !




 


! VL6
AAT TTT ATG CTG ACT CAG CCC CAC TCT GTG TCG GAG
6a
(SEQ ID NO: 340)



TCT CCG GGG AAG ACG GTA ACC ATC TCC TGC !




 


! VL7
CAG ACT GTG GTG ACT CAG GAG CCC TCA CTG ACT GTG
7a
(SEQ ID NO: 341)



TCC CCA GGA GGG ACA GTC ACT CTC ACC TGT !




 



cag Gct gtg gtg act cag gag ccc tca ctg act gtg
7b
(SEQ ID NO: 342)



tcc cca gga ggg aca gtc act ctc acc tgt !




 


! VL8
CAG ACT GTG GTG ACC CAG GAG CCA TCG TTC TCA GTG
8a
(SEQ ID NO: 343)



TCC CCT GGA GGG ACA GTC ACA CTC ACT TGT !




 


! VL9
CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA TCA GCC
9a
(SEQ ID NO: 344)



TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC !




 


! VL10
CAG GCA GGG CTG ACT CAG CCA CCC TCG GTG TCC AAG
10a
(SEQ ID NO: 345)



GGC TTG AGA CAG ACC GCC ACA CTC ACC TGC !
















TABLE 11 





RERSs found in human lambda FR1 GLGs


! There are 31 lambda GLGs
















MlyI NnnnnnGACTC (SEQ ID NO: 346)
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 (SEQ ID NO: 347)
 1







26: 34








MwoI GCNNNNNnngc (SEQ ID NO: 348) 
20







 1:  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








HinfI 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








PleI 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








DdeI 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.








MnlI 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
 7







 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








MaeIII 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 (SEQ ID NO: 349)
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


 


AF103099
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



 


af103286
HSA235677
HSU94412
MCOMFRVA



 


AF103309
HSA235676
HSU94415
S82745



 


af103343
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
Seq1
gtgtattactgtgc
25


 


2
19
 7
 6
 4
 2
 0
Seq2
atAtattactgtgc
26


 


3
 1
 0
 0
 1
 0
 0
Seq3
gtgtattactgtAA
27


 


4
 7
 1
 5
 1
 0
 0
Seq4
gtgtattactgtAc
28


 


5
 0
 0
 0
 0
 0
 0
Seq5
Ttgtattactgtgc
29


 


6
 0
 0
 0
 0
 0
 0
Seq6
TtgtatCactgtgc
30


 


7
 3
 1
 0
 1
 1
 0
Seq7
ACAtattactgtgc
31


 


8
 2
 0
 2
 0
 0
 0
Seq8
ACgtattactgtgc
32


 


9
 9
 2
 2
 4
 1
 0
Seq9
ATgtattactgtgc
33















Group
26
26
21
 4
 2





Cumulative
26
52
73
77
79










Table 12C Most important URE recognition seqs in FR3 Heavy














1
VHSzy1
GTGtattactgtgc
(ON_SHC103)
(SEQ ID NO: 25)


 


2
VHSzy2
GTAtattactgtgc
(ON_SHC323)
(SEQ ID NO: 26)


 


3
VHSzy4
GTGtattactgtac
(ON_SHC349)
(SEQ ID NO: 28)


 


4
VHSzy9
ATGtattactgtgc
(ON_SHC5a)
(SEQ ID NO: 33)










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


Number of sequences..........    79


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

















Number of mismatches




















Id
Best
0
1
2
3
4
5








1
39
15
11
10
 1
 2
0
Seq1
gtgtattactgtgc
(SEQ ID NO: 25)


 


2
22
 7
 6
 5
 3
 0
1
Seq2
gtAtattactgtgc
(SEQ ID NO: 26)


 


3
 7
 1
 5
 1
 0
 0
0
Seq4
gtgtattactgtAc
(SEQ ID NO: 28)


 


4
11
 2
 4
 4
 1
 0
0
Seq9
ATgtattactgtgc
(SEQ ID NO: 33)
















Group
25
26
20
 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 VHSzyl1 (ID = 1) 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/cgi-bin/asymmlist.


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





AarI
CACCTGCNNNN{circumflex over ( )}NNNN_
—
y


 


AceIII
CAGCTCNNNNNNN{circumflex over ( )}NNNN_
—
—


 


Bbr7I
GAAGACNNNNNNN{circumflex over ( )}NNNN_
—
—


 


BbvI
GCAGCNNNNNNNN{circumflex over ( )}NNNN_

y


 


BbvII
GAAGACNN{circumflex over ( )}NNNN_




 


Bce83I
CTTGAGNNNNNNNNNNNNNN_NN{circumflex over ( )}
—
—


 


BceAI
ACGGCNNNNNNNNNNNNN{circumflex over ( )}NN_
—
y


 


BcefI
ACGGCNNNNNNNNNNNNN{circumflex over ( )}N_
—
—


 


BciVI
GTATCCNNNNN_N{circumflex over ( )} 
BfuI
y


 


BfiI
ACTGGGNNNN_N{circumflex over ( )}
BmrI
y


 


BinI
GGATCNNNN{circumflex over ( )}N_




 


BscAI
GCATCNNNN{circumflex over ( )}NN_
—
—


 


BseRI
GAGGAGNNNNNNNN_NN{circumflex over ( )}
—
y


 


BsmFI
GGGACNNNNNNNNNN{circumflex over ( )}NNNN_
BspLU11III
y


 


BspMI
ACCTGCNNNN{circumflex over ( )}NNNN_ 
Acc36I
y


 


EciI
GGCGGANNNNNNNNN_NN{circumflex over ( )}
—
y


 


Eco57I
CTGAAGNNNNNNNNNNNNNN_NN{circumflex over ( )}
BspKT5I
y


 


FauI
CCCGCNNNN{circumflex over ( )}NN_
BstFZ438I
y


 


FokI
GGATGNNNNNNNNN{circumflex over ( )}NNNN_
BstPZ418I
y


 


GsuI
CTGGAGNNNNNNNNNNNNNN_NN{circumflex over ( )}
—
y


 


HgaI
GACGCNNNNN{circumflex over ( )}NNNNN_
—
y


 


HphI
GGTGANNNNNNN_N{circumflex over ( )}
AsuHPI
y


 


MboII
GAAGANNNNNNN_N{circumflex over ( )}
—
y


 


MlyI
GAGTCNNNNN{circumflex over ( )}
SchI
y


 


MmeI
TCCRACNNNNNNNNNNNNNNNNNN_NN{circumflex over ( )}
—
—


 


MnlI
CCTCNNNNNN_N{circumflex over ( )}
—
y


 


PleI
GAGTCNNNN{circumflex over ( )}N_
PpsI
y


 


RleAI
CCCACANNNNNNNNN_NNN{circumflex over ( )}
—
—


 


SfaNI
GCATCNNNNN{circumflex over ( )}NNNN_
BspST5I
y


 


SspD5I
GGTGANNNNNNNN{circumflex over ( )}
—
—


 


Sth132I
CCCGNNNN{circumflex over ( )}NNNN_
—
—


 


StsI
GGATGNNNNNNNNNNNN{circumflex over ( )}NNNN_
—
—


 


TaqII
GACCGANNNNNNNNN_NN{circumflex over ( )},
—
—


 



CACCCANNNNNNNNN_NN{circumflex over ( )}




 


Tth111II
CAARCANNNNNNNNN_NN{circumflex over ( )}
—
—


 


UbaPI
CGAACG
—
—


 







(SEQ ID NOS 356-390, respectively in order of appearance)





The notation is {circumflex over ( )} 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 {circumflex over ( )} appears.













TABLE 14







(FOKIact)       5′-cAcATccgTg TTgTT cAcggATgTg-3′ (SEQ ID NO: 350)


(VHEx881) 5′-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-


                        AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3′ (SEQ ID NO: 351)


! note that VHEx881 is the reverse complement of the ON below








 








(VHBA881)
            





 


(VHBB881)
            





 


(VH881PCR)




















TABLE 15





Use of FokI as “Universal Restriction Enzyme”















FokI - for dsDNA, ∥ represents sites of cleavage


 


         





 


Case I


 


                        





 


Case II


 


          





 


Case III (Case I rotated 180 degrees)


 


          





 


Case IV (Case II rotated 180 degrees)


 


 





 


Improved FokI adapters


FokI - for dsDNA, ∥ represents sites of cleavage


Case I


Stem 11, loop 5, stem 11, recognition 17


 


                                 





 


Case II


Stem 10, loop 5, stem 10, recognition 18


 


          





 


Case III (Case I rotated 180 degrees)


Stem 11, loop 5, stem 11, recognition 20


 


            





 


Case IV (Case II rotated 180 degrees)


Stem 11, loop 4, stem 11, recognition 17


 


 





 


BseRI


 


           





 


Stem 11, loop 5, stem 11, recognition 19


 






















TABLE 16





Human heavy chains bases 88.1 to 94.2















Number of sequences . . . . . . . . . . 840












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































Probe



Id
Ntot
0
1
2
3
4
5
6
7
Name
Sequence............
Dot form............





1
364
152
 97
 76
 26
  7
  4
  2
  0
VHS881-
gctgtgtattactgtgcgag
gctgtgtattactgtgcgag












1.1




2
265
150
 60
 33
 13
  5
  4
  0
  0
VHS881-
gccgtgtattactgtgcgag
..c.................












1.2




3
 96
 14
 34
 16
 10
  5
  7
  9
  1
VHS881-
gccgtatattactgtgcgag
..c..a..............












2.1




4
 20
 0
  3
  4 
  9
  2
  2
  0
  0
VHS881-
gccgtgtattactgtacgag
..c............a....












4.1




5
 95
 25
 36
 18
 11
  2
  2
  0
  1
VHS881-
gccatgtattactgtgcgag
..ca................












9.1




















840
341
230
147
 69
 21
 19
 11
  2
(SEQ ID NOS 391-395, respectively in order of




341
571
718
787
808
827
838
840
appearance)
















 


(Sequences in the left column above are SEQ ID NOS 391-395, respectively in order of


appearance; sequences in the right column are all SEQ ID NO: 396)


(FOKIact) 5′-cAcATccgTg TTgTT cAcggagTg-3′ (SEQ ID NO: 396)


(VHEx881) 5′-AATAgTAgAc TgcAgTgTcc TcAgcccTTA AgcTgTTcAT cTgcAAgTAg-


       AgAgTATTcT TAgAgTTgTc TcTAgAcTTA gTgAAgcg-3′ (SEQ ID NO: 397)


! note that VHEx881 is the reverse complement of the ON below


 




















(VHBA881)






 


(VHBB881)






 


(VH881PCR)




















TABLE 17





Kappa, bases 12-30




























!















!
ID
Ntot
0
 1
 2
 3
 4
 5
 6
Name
Sequence...........
Dot Form...........





!
1
 84   
40
 21
 20
  1
  2
  0
  0
SK12O12
gacccagtctccatcctcc
gacccagtctccatcctcc
(residues 26-44 of















SEQ ID NO: 400)


 


!
2
 32
19
  3
  6
  2
  1
  0
  1
SK12A17
gactcagtctccactctcc
...t.........ct....
(residues 26-44 of















SEQ ID NO: 401)


 


!
3
 26
17
  8
  1
  0
  0
  0
  0
SK12A27
gacgcagtctccaggcacc
...g.........gg.a..
(residues 26-44 of















SEQ ID NO: 402)


 


!
4
 40
21
 18
  1
  0
  0
  0
  0
SK12A11
gacgcagtctccagccacc
...g.........g..a..
(residues 26-44 of















SEQ ID NO: 403)


 


!

182
97
 50
 28
  3
  3 
  0
  1






!

 
97
147
175
178
181
181
182






!










URE adapters:


!                Stem...... Loop. Stem...... Recognition.......


(SzKB1230-O12)           5′-cAcATccgTg TTgTT cAcggATgTg ggAggATggAgAcTgggTc-3′ (SEQ ID NO: 400)


!            [RC] 5′-gacccagtctccatcctcc cAcATccgTg AAcAA cAcggATgTg-3′


!                Recognition........ Stem...... loop. Stem......


!                                 FokI.            FokI.


!


 


!                Stem...... Loop. Stem...... Recognition........


(SzKB1230-A17)           5′-cAcATccgTg TTgTT cAcggATgTg ggAgAgTggAgAcTgAgTc-3′ (SEQ ID NO: 401)


!            [RC] 5′-gactcagtctccactctcc cAcATccgTg AAcAA cAcggATgTg-3′


!                Recognition........ Stem...... loop. Stem......


!                                  FokI.           FokI.


!


 


!                Stem...... Loop. Stem...... Recognition........


(SzKB1230-A27)           5′-cAcATccgTg TTgTT cAcggATgTg ggTgccTggAgAcTgcgTc-3′ (SEQ ID NO: 402)


!            [RC] 5′-gacgcagtctccaggcacc cAcATccgTg AAcAA cAcggATgTg-3′


!                Recognition........ Stem...... loop. Stem......


!                                  FokI.           FokI.


!


 


!                Stem...... Loop. Stem....... Recognition........


(SzKB1230-A11)           5′-cAcATccgTg TTgTT cAcggATgTg ggTggcTggAgAcTgcgTc-3′ (SEQ ID NO: 403)


!            [RC] 5′-gacgcagtctccagccacc cAcATccgTg AAcAA cAcggATgTg-3′


!                Recognition........ Stem...... loop. Stem......


!                                  FokI.            FokI.


 


What happens in the upper strand:


 


(SzKB1230-O12*)    5′-gac cca gtc | tcc a-tc ctc c-3′  (residues 26-44 of SEQ ID NO: 400)








!
                  | Site of cleavage in substrate


!








(SzKB1230-A17*)    5′-gac tca gtc | tcc a-ct ctc c-3′  (residues 26-44 of SEQ ID NO: 401)








!








(SzKB1230-A27*)    5′-gac gca gtc | tcc a-gg cac c-3′  (residues 26-44 of SEQ ID NO: 402)








!








(SzKB1230-A11*)    5′-gac gca gtc | tcc a-gc cac c-3′  (residues 26-44 of SEQ ID NO: 403)


 


(kapextURE)     5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg-3′ !sense strand (SEQ ID NO: 404)


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


 


(kapextUREPCR)  5′-ccTctactctTgTcAcAgTg-3′ (SEQ ID NO: 405)


                   Scab.............


 


(kaBR01UR)   5′-ggAggATggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′       (SEQ ID NO: 406)


!        [RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-tc ctc c-3′   ON above is R.C. of this one


 


(kaBR02UR)   5′-ggAgAgTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′       (SEQ ID NO: 407)


!        [RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-ct ctc c-3′   ON above is R.C. of this one


 


(kaBR03UR)   5′-ggTgccTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′       (SEQ ID NO: 408)


!        [RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gg cac c-3′   ON above is R.C. of this one


 


(kaBR04UR)   5′-ggTggcTggA cTggATgTcT TgTgcAcTgT gAcAAgAgTA gAgg-3′       (SEQ ID NO: 409)


!        [RC] 5′-ccTctactctTgTcAcAgTgcAcAA gAc ATc cAg tcc a-gc cac c-3′   ON above is R.C. of this one


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
















TABLE 18 





Lambda URE adapters bases 13.3 to 19.3















!


! Number of sequences.......... 128


!









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





















! Id
Ntot
0
1
2
3
4
5
6
7
8
Name
Sequence...........
Dot form...........





! 1
 58
45
 7
 1
 0
 0
  0
  2
  2
  1
VL133-
gtctcctggacagtcgatc
gtctcctggacagtcgatc













2a2

(residues 632-635















of SEQ ID NO: 410)


! 2
 16
10
 1
 0
 1
 0
  1
  1
  0
  2
VL133-
ggccttgggacagacagtc
.g.cttg......a.ag..













31

(residues 632-635















of SEQ ID NO: 411)


! 3
 17
 6
 0
 0
 0
 4
  1
  1
  5
  0
VL133-
gtctcctggacagtcagtc
...............ag..













2c

(residues 632-635















of SEQ ID NO: 412)


! 4
 37
 3
 0
10
 4
 4
  3
  7
  4
  2
VL133-
ggcccagggcagagggtc
.g.c..a..g...ag.g..













1c

(residues 632-635















of SEQ ID NO: 413)


! 
128
64
 8 
11
 5
 8
  5 
 11 
 11
  5





! 

64
72
83
88
96
101
112
123
128










! 


!           Stem...... loop. Stem...... Recognition........


(VL133-2a2)     5′-cAcATccgTg TTgTT cAcggATgTg gATcgAcTgTccAggAgAc-3′ (SEQ ID NO: 410)


!      [RC] 5′-gtctcctggacagtcgatc cAcATccgTg AAcAA cAcggATgTg-3′


!           Recognition........ Stem...... Loop. Stem......


!           Stem...... loop. Stem...... Recognition........


(VL133-31)     5′-cAcATccgTg TTgTT cAcggATgTg gAcTgTcTgTcccAAggcc-3′ (SEQ ID NO: 411)


!      [RC] 5′-ggccttgggacagacagtc cAcATccgTg AAcAA cAcggATgTg-3′


!           Recognition........ Stem...... Loop. Stem......


!


!           Stem...... loop. Stem...... Recognition........


(VL133-2c)     5′-cAcATccgTg TTgTT cAcggATgTg gAcTgAcTgTccAggAgAc-3′ (SEQ ID NO: 412)


       [RC] 5′-gtctcctggacagtcagtc cAcATccgTg AAcAA cAcggATgTg-3′


!           Recognition........ Stem...... Loop. Stem......


!


!           Stem...... loop. Stem...... Recognition........


(VL133-1c)     5′-cAcATccgTg TTgTT cAcggATgTg gAcccTcTgcccTggggcc-3′ (SEQ ID NO: 413)


       [RC] 5′-ggccccagggcagagggtc cAcATccgTg AAcAA cAcggATgTg-3′


What happens in the top strand:


 








! (VL133-2a2*)






 


! (VL133-31*) 






 


! (VL133-2c*) 






 


! (VL133-1c*)






 







! 


! The following Extenders and Bridges all encode the AA sequence of 2a2 for codons 1-15


!                    1


(ON_LamEx133) 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-


!


!    13  14  15


     tcC ccG g ! 2a2 (SEQ ID NO: 414)


!                     1


(ON_LamB1-133) [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-


! 


!     13 14 15


     tcC ccG g ga cag tcg at-3′ !(SEQ ID NO: 415) 2a2 N.B. the actual seq is the


!                          reverse complement of the


!                          one shown.


! 


(ON_LamB2-133) [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-


! 


!     13 14 15


     tcC ccG g ga cag aca gt-3′ ! 31 (SEQ ID NO: 416) N.B. the actual seq is the


!                          reverse complement of the


!                          one shown.


! 


! 


(ON_LamB3-133) [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-


! 


!    13 14 15


    tcC ccG g ga cag tca gt -3′! 2c (SEQ ID NO: 417) N.B. the actual seq is the


!                         reverse complement of the


!                         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


!


!     13  14  15


     tcC ccG g gg cag agg gt-3′ ! 1c (SEQ ID NO: 418) N.B. the actual seq is the


!                                      reverse complement of the


!                                      one shown.


!


(ON_Lam133PCR) 5′-ccTcTgAcTgAgT gcA cAg AGt gc-3′ (SEQ ID NO: 419)
















TABLE 19







Cleavage of 75 human light chains.















Planned


Enzyme
Recognition*
Nch
Ns
location of site














AfeI
AGCgct
0
0



AflII
Cttaag
0
0
HC FR3


AgeI
Accggt
0
0



AscI
GGcgcgcc
0
0
After LC


BglII
Agatct
0
0



BsiWI
Cgtacg
0
0



BspDI
ATcgat
0
0



BssHII
Gcgcgc
0
0



BstBI
TTcgaa
0
0



DraIII
CACNNNgtg
0
0



EagI
Cggccg
0
0



FseI
GGCCGGcc
0
0



FspI
TGCgca
0
0



HpaI
GTTaac
0
0



MfeI
Caattg
0
0
HC FR1


MluI
Acgcgt
0
0



NcoI
Ccatgg
0
0
Heavy chain signal


NheI
Gctagc
0
0
HC/anchor linker


NotI
GCggccgc
0
0
In linker after HC


NruI
TCGcga
0
0



PacI
TTAATtaa
0
0



PmeI
GTTTaaac
0
0



PmlI
CACgtg
0
0



PvuI
CGATcg
0
0



SacII
CCGCgg
0
0



SalI
Gtcgac
0
0



SfiI
GGCCNNNNnggcc
0
0
Heavy Chain signal






(SEQ ID NO: 436)


SgfI
GCGATcgc
0
0



SnaBI
TACgta
0
0



StuI
AGGcct
0
0



XbaI
Tctaga
0
0
HC FR3


AatII
GACGTc
1
1



AclI
AAcgtt
1
1



AseI
ATtaat
1
1



BsmI
GAATGCN
1
1



BspEI
Tccgga
1
1
HC FR1






(SEQ ID NO: 437)


BstXI
CCANNNNNntgg
1
1
HC FR2






(SEQ ID NO: 438)


DrdI
GACNNNNnngtc
1
1



HindIII
Aagctt
1
1



PciI
Acatgt
1
1



SapI
gaagagc
1
1



ScaI
AGTact
1
1



SexAI
Accwggt
1
1



SpeI
Actagt
1
1



TliI
Ctcgag
1
1



XhoI
Ctcgag
1
1



BcgI
cgannnnnntgc
2
2
(SEQ ID NO: 439)


BlpI
GCtnagc
2
2



BssSI
Ctcgtg
2
2



BstAPI
GCANNNNntgc
2
2
(SEQ ID NO: 440)


EspI
GCtnagc
2
2



KasI
Ggcgcc
2
2



PflMI
CCANNNNntgg
2
2
(SEQ ID NO: 441)


XmnI
GAANNnnttc
2
2
(SEQ ID NO: 442)


ApaLI
Gtgcac
3
3
LC signal seq


NaeI
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



BclI
Tgatca
5
5



BsmBI
Nnnnnngagacg
5
5
(SEQ ID NO: 443)


BsrGI
Tgtaca
5
5



DraI
TTTaaa
6
6



NdeI
CAtatg
6
6
HC FR4


SwaI
ATTTaaat
6
6



BamHI
Ggatcc
7
7



SacI
GAGCTc
7
7



BciVI
GTATCCNNNNNN
8
8
(SEQ ID NO: 444)


BsaBI
GATNNnnatc
8
8
(SEQ ID NO: 619)


NsiI
ATGCAt
8
8



Bsp120I
Gggccc
9
9
CH1


ApaI
GGGCCc
9
9
CH1


PspOOMI
Gggccc
9
9



BspHI
Tcatga
9
11



EcoRV
GATatc
9
9



AhdI
GACNNNnngtc
11
11
(SEQ ID NO: 445)


BbsI
GAAGAC
11
14



PsiI
TTAtaa
12
12



BsaI
GGTCTCNnnnn
13
15
(SEQ ID NO: 446)


XmaI
Cccggg
13
14



AvaI
Cycgrg
14
16



BglI
GCCNNNNnggc
14
17
(SEQ ID NO: 447)


AlwNI
CAGNNNctg
16
16



BspMI
ACCTGC
17
19



XcmI
CCANNNNNnnnntgg
17
26
(SEQ ID NO: 448)


BstEII
Ggtnacc
19
22
HC FR4


Sse8387I
CCTGCAgg
20
20



AvrII
Cctagg
22
22



HincII
GTYrac
22
22



BsgI
GTGCAG
27
29



MscI
TGGcca
30
34



BseRI
NNnnnnnnnnctcctc
32
35
(SEQ ID NO: 449)


Bsu36I
CCtnagg
35
37



PstI
CTGCAg
35
40



EciI
nnnnnnnnntccgcc
38
40
(SEQ ID NO: 450)


PpuMI
RGgwccy
41
50



StyI
Ccwwgg
44
73



Eco0109I
RGgnccy
46
70



Acc65I
Ggtacc
50
51



KpnI
GGTACc
50
51



BpmI
ctccag
53
82



AvaII
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















Planned


Enzyme
Recognition
Nch
Ns
location of site














AfeI
AGCgct
0
0



AflII
Cttaag
0
0
HC FR3


AscI
GGcgcgcc
0
0
After LC


BsiWI
Cgtacg
0
0



BspDI
ATcgat
0
0



BssHII
Gcgcgc
0
0



FseI
GGCCGGcc
0
0



HpaI
GTTaac
0
0



NheI
Gctagc
0
0
HC Linker


NotI
GCggccgc
0
0
In linker, HC/anchor


NruI
TCGcga
0
0



NsiI
ATGCAt
0
0



PacI
TTAATtaa
0
0



PciI
Acatgt
0
0



PmeI
GTTTaaac
0
0



PvuI
CGATcg
0
0



RsrII
CGgwccg
0
0



SapI
gaagagc
0
0



SfiI
GGCCNNNNnggcc
0
0
HC signal seq






(SEQ ID NO: 420)


SgfI
GCGATcgc
0
0



SwaI
ATTTaaat
0
0



AclI
AAcgtt
1
1



AgeI
Accggt
1
1



AseI
ATtaat
1
1



AvrII
Cctagg
1
1



BsmI
GAATGCN
1
1



BsrBI
GAGcgg
1
1



BsrDI
GCAATGNNn
1
1



DraI
TTTaaa
1
1



FspI
TGCgca
1
1



HindIII
Aagctt
1
1



MfeI
Caattg
1
1
HC FR1


NaeI
GCCggc
1
1



NgoMI
Gccggc
1
1



SpeI
Actagt
1
1



Acc65I
Ggtacc
2
2



BstBI
TTcgaa
2
2



KpnI
GGTACc
2
2



MluI
Acgcgt
2
2



NcoI
Ccatgg
2
2
In HC signal seq


NdeI
CAtatg
2
2
HC FR4


PmlI
CACgtg
2
2



XcmI
CCANNNNNnnnntgg
2
2
(SEQ ID NO: 421)


BcgI
cgannnnnntgc
3
3
(SEQ ID NO: 422)


BclI
Tgatca
3
3



BglI 
GCCNNNNnggc
3
3
(SEQ ID NO: 423)


BsaBI
GATNNnnatc
3
3
(SEQ ID NO: 424)


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



PsiI
TTAtaa
4
5



SphI
GCATGc
4
4



AhdI
GACNNNnngtc
5
5
(SEQ ID NO: 425)


BspEI
Tccgga
5
5
HC FR1


MscI
TGGcca
5
5



SacI
GAGCTc
5
5



ScaI
AGTact
5
5



SexAI
Accwggt
5
6



SspI
AATatt
5
5



TliI
Ctcgag
5
5



XhoI
Ctcgag
5
5



BbsI
GAAGAC
7
8



BstAPI
GCANNNNntgc
7
8
(SEQ ID NO: 426)


BstZ17I
GTAtac
7
7



EcoRV
GATatc
7
7



EcoRI
Gaattc
8
8



BlpI
GCtnagc
9
9



Bsu36I
CCtnagg
9
9



DraIII
CACNNNgtg
9
9



EspI
GCtnagc
9
9



StuI
AGGcct
9
13



XbaI
Tctaga
9
9
HC FR3


Bsp120I
Gggccc
10
11
CH1


ApaI
GGGCCc
10
11
CH1


PspOOMI
Gggccc
10
11



BciVI
GTATCCNNNNNN
11
11
(SEQ ID NO: 427)


SalI
Gtcgac
11
12



DrdI
GACNNNNnngtc
12
12
(SEQ ID NO: 428)


KasI
Ggcgcc
12
12



XmaI
Cccggg
12
14



BglII
Agatct
14
14



HincII
GTYrac
16
18



BamHI
Ggatcc
17
17



Pf1MI
CCANNNNntgg
17
18
(SEQ ID NO: 429)


BsmBI
Nnnnnngagacg
18
21
(SEQ ID NO: 430)


BstXI
CCANNNNNntgg
18
19
HC FR2






(SEQ ID NO: 431)


XmnI
GAANNnnttc
18
18
(SEQ ID NO: 432)


SacII
CCGCgg
19
19



PstI
CTGCAg
20
24



PvuII
CAGctg
20
22



AvaI
Cycgrg
21
24



EagI
Cggccg
21
22



AatII
GACGTc
22
22



BspMI
ACCTGC
27
33



AccI
GTmkac
30
43



StyI
Ccwwgg
36
49



AlwNI
CAGNNNctg
38
44



BsaI
GGTCTCNnnnn
38
44
(SEQ ID NO: 433)


PpuMI
RGgwccy
43
46



BsgI
GTGCAG
44
54



BseRI
NNnnnnnnnnctcctc 
48
60
(SEQ ID NO: 434)


EciI
nnnnnnnnntccgcc
52
57
(SEQ ID NO: 435)


BstEII
Ggtnacc
54
61
HC Fr4,47/79






have one


Eco0109I
RGgnccy
54
86



BpmI
ctccag
60
121



AvaII
Ggwcc
71
140
















TABLE 21





MALIA3, annotated















! MALIA3 9532 bases


!----------------------------------------------------------------------


    (SEQ ID NO: 451)


     1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc


!    gene ii continued


    49 cca aat gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta


    97 tct aat ggt caa act aaa tct act cgt tcg cag aat tgg gaa tca act


   145 gtt aca tgg aat gaa act tcc aga cac cgt act tta gtt gca tat tta


   193 aaa cat gtt gag cta cag cac cag att cag caa tta agc tct aag cca


   241 tcc gca aaa atg acc tct tat caa aag gag caa tta aag gta ctc tct


   289 aat cct gac ctg ttg gag ttt gct tcc ggt ctg gtt cgc ttt gaa gct


   337 cga att aaa acg cga tat ttg aag tct ttc ggg ctt cct ctt aat ctt


   385 ttt gat gca atc cgc ttt gct tct gac tat aat agt cag ggt aaa gac


   433 ctg att ttt gat tta tgg tca ttc tcg ttt tct gaa ctg ttt aaa gca


   481 ttt gag ggg gat tca ATG aat att tat gac gat tcc gca gta ttg gac


!          RBS?......   Start gene x, ii continues


   529 gct atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct


   577 ttt gca aaa gcc tct cgc tat ttt ggt ttt tat cgt cgt ctg gta aac


   625 gag ggt tat gat agt gtt gct ctt act atg cct cgt aat tcc ttt tgg


   673 cgt tat gta tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg


   721 atg aat ctt tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att


   769 aac gta gat ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt


   817 ctt aaa atc gca TAA


!                      End X & II


   832 ggtaattca ca


!


   (SEQ ID NO: 623)


!       M1              E5                 Q10                 T15


   843 ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt


!      Start gene V


!


!      S17         S20                 P25                 E30


   891 tct ggt gtt tct cgt cag ggc aag cct tat tca ctg aat gag cag ctt


!


!              V35                 E40                 V45


   939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ctt gtc aag att act


!


!          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


!                      P85     K87 end of V


  1083 ctg cgc ctc gtt ccg gct aag TAA C


!


  1108 ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg


!      Start gene VII


!


  1150 ata caa atc tcc gtt gta ctt tgt ttc gcg ctt ggt ata atc


!


!                        VII and IX overlap.


!                        ..... S2 V3 L4 V5 (SEQ ID NO: 624)         S10


  1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttc gcc tct ttc gtt


 








 


!


!       .... stop of IX, IX and VIII overlap by four bases


  1301 ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct acc ctc


!      Start signal sequence of viii.


!


  1349 gtt ccg atg ctg tct ttc gct gct gag ggt gac gat ccc gca aaa gcg


!                                  mature VIII --->


  1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat gcg


  1445 tgg gcg atg gtt gtt gtc att


  1466 gtc ggc gca act atc ggt atc aag ctg ttt aag


  1499 aaa ttc acc tcg aaa gca ! 1515


!       ...........  −35  ..


!


  1517      agc tga taaaccgat acaattaaag gctccttttg


!                      ..... −10   ...


!


  1552 gagccttttt ttttGGAGAt ttt ! S.D. underlined


!


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


!            M   K   K   L   L   F   A   I   P   L   V (SEQ ID NO: 452)


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


!


!       V   P   F   Y   S   H   S   A   Q


  1612 gtt cct ttc tat tct cac aGT gcA Cag tCT


!                               ApaLI...


!


  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


  2146 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.....


!     (SEQ ID NO: 453)


!      Pe1B signal ----------------------------------------------------->


!       M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L


  2343 ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC


!


!       16  17  18  19  20     21 22


!       A   A   Q   P   A       M   A


  2388 gcG GCC cag ccG GCC     atg gcc


!        SfiI.............


!                NgoMI...(1/2)


!                       NcoI........


!


 




















!BstXI.
































 





! From BstEII onwards, pV323 is same as pCES1, except as noted.


! 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


!                          A   S   T   K   G   P   S   V   F   P


  2769                    gcc tcc acc aaG GGC CCa tcg GTC TTC ccc


!                                       Bsp120I.      BbsI...(2/2)


!                                       ApaI....


!      153 154 155 156 157 158 159 160 161 162 163 164 165 166 167


!       L   A   P   S   S   K   S   T   S   G   G   T   A   A   L


  2799 ctg gca ccC TCC TCc aag agc acc tct ggg ggc aca gcg gcc ctg


!                                       BseRI...(2/2)


!        168 169 170 171 172 173 174 175 176 177 178 179 180 181 182


!         G   C   L   V   K   D   Y   F   P   E   P   V   T   V   S


  2844   ggc tgc ctg GTC AAG GAC TAC TTC CCc gaA CCG GTg acg gtg tcg


!                                              AgeI....


!        183 184 185 186 187 188 189 190 191 192 193 194 195 196 197


!         W   N   S   G   A   L   T   S   G   V   H   T   F   P   A


  2889   tgg aac tca GGC GCC ctg acc agc ggc gtc cac acc ttc ccg get


!                    KasI...(1/4)


!


!        198 199 200 201 202 203 204 205 206 207 208 209 210 211 212


!         V   L   Q   S   S   G   L   Y   S   L   S   S   V   V   T


  2934   gtc cta cag tCt agc GGa ctc tac tcc ctc agc agc gta gtg acc


!                    (Bsu36I...)(knocked out)


!        213 214 215 216 217 218 219 220 221 222 223 224 225 226 227


!         V   P   S   S   S   L   G   T   Q   T   Y   I   C   N   V


  2979   gtg ccC tCt tct agc tTG Ggc acc cag acc tac atc tgc aac gtg


!                (BstXI ..........)N.B. destruction of BstXI & BpmI sites.


!


!        228 229 230 231 232 233 234 235 236 237 238 239 240 241 242


!         N   H   K   P   S   N   T   K   V   D   K   K   V   E   P


  3024   aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc


!


!        243 244 245


!         K   S   C   A   A   A   H   H   H   H   H   H   S   A


  3069   aaa tct tgt GCG GCC GCt cat cac cac cat cat cac tct gct


!                    NotI......


!         E   Q   K   L   I   S   E   E   D   L   N   G   A   A


  3111   gaa caa aaa ctc atc tca gaa gag gat ctg aat ggt gcc gca


!


!


!         D   I   N   D   D   R   M     A   S   G   A


  3153   GAT ATC aac gat gat cgt atg   gct AGC ggc gcc


!        rEK cleavage site..........   NheI... KasI...


!        EcoRV..


!


! Domain 1 ---------------------------------------------------------------


!           A   E   T   V   E   S   C   L   A


  3183     gct gaa act gtt gaa agt tgt tta gca


!


!


!       K   P   H   T   E   N   S   F


  3210 aaa ccc cat aca gaa aat tca ttt


! 


!       T   N   V   W   K   D   D   K   T


  3234 aCT AAC GTC TGG AAA GAC GAC AAA Act


!


!       L   D   R   Y   A   N   Y   E   G   C   L   W   N   A   T   G   V


  3261 tta gat cgt tac gct aac tat gag ggt tgt ctg tgG AAT GCt aca ggc gtt


!                                                    BsmI____


!


!       V   V   C   T   G   D   E   T   Q   C   Y   G   T   W   V   P   I


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


!


!       G   L   A   I   P   E   N


  3363 ggg ctt gct atc cct gaa aat


!


! L1 linker ------------------------------------


!       E   G   G   G   S   E   G   G   G   S


  3384 gag ggt ggt ggc tct gag ggt ggc ggt tct


!


!       E   G   G   G   S   E   G   G   G   T


  3414 gag ggt ggc ggt tct gag ggt ggc ggt act


!


!  Domain 2 ------------------------------------


  3444 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat atc aac


  3495 cct ctc gac ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct


  3546 aat cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag aat


!                      BseRI__


  3597 aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc act


  3645 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct


  3693 gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt aaa ttC AGA


!                                                                AlwNI


  3741 GAC TGc gct ttc cat tct ggc ttt aat gaa gat cca ttc gtt tgt gaa


!       AlwNI


  3789 tat caa ggc caa tcg tct gac ctg cct caa cct cct gtc aat gct


!


  3834 ggc ggc ggc tct


!  start L2


-------------------------------------------------------------


  3846 ggt ggt ggt tct


  3858 ggt ggc ggc tct


  3870 gag ggt ggt ggc tct gag ggt ggc ggt tct


  3900 gag ggt ggc ggc tct gag gga ggc ggt tcc


  3930 ggt ggt ggc tct ggt    ! end L2


!


! Domain 3


(SEQ ID NO: 454)


--------------------------------------------------------------


!       S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A


  3945 tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct


!


!       M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G


  3993 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc


!


!       K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F


  4041 aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc


!


!       I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D


  4089 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat


!


!       F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N


  4137 ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat


!


!       S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q


  4185 tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa


!


!       S   V   E   C   R   P   F   V   F   S   A   G   K   P   Y   E


  4233 tcg gtt gaa tgt cgc cct ttt gtc ttt agc gct ggt aaa cca tat gaa


!


!       F   S   I   D   C   D   K   I   N   L   F   R


  4281 ttt tct att gat tgt gac aaa ata aac tta ttc cgt


!                                                  End Domain 3


!


!       G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V  F140


  4317 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt


!      start transmembrane segment


!     


!       S   T   F   A   N   I   L


  4365 tct acg ttt gct aac ata ctg


!


!       R   N   K   E   S


  4386 cgt aat aag gag tct TAA ! stop of iii


!     Intracellular anchor.


!


          (SEQ ID NO: 455)


!           M1  P2  V   L  L5   G   I   P   L  L10  L   R  FL  G15


  4404  tc ATG cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc ctc ggt


!          Start VI


!


  4451 ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag


  4499 ggc ttc ggt aag ata gct att gct att tca ttg ttt ctt gct ctt att


  4547 att ggg ctt aac tca att ctt gtg ggt tat ctc tct gat att agc gct


  4595 caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att ctc ccg tct


  4643 aat gcg ctt ccc tgt ttt tat gtt att ctc tct gta aag gct gct att


  4691 ttc att ttt gac gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat


!


                 (SEQ ID NO: 456)


!                 M1  A2  V3      F5                 L10         G13


  4739 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga


!       end VI Start gene I


!       14  15  16  17  18  19  20  21  22  23  24  25  26  27  28


!       K   T   L   V   S   V   G   K   I   Q   D   K   I   V   A


  4785 aag acg ctc gtt agc gtt ggt aag att cag gat aaa att gta gct


!


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


!       G   C   K   I   A   T   N   L   D   L   R   L   Q   N   L


  4830 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac ctc


!


        44  45  46  47  48  49  50  51  52  53  54  55  56  57  58


!       P   Q   V   G   R   F   A   K   T   P   R   V   L   R   I


  4875 ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata


!


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


!       P   D   K   P   S   I   S   D   L   L   A   I   G   R   G


  4920 ccg gat aag cct tct ata tct gat ttg ctt gct att ggg cgc ggt


!


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


!       N   D   S   Y   D   E   N   K   N   G   L   L   V   L   D


  4965 aat gat tcc tac gat gaa aat aaa aac ggc ttg ctt gtt ctc gat


!


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


!       E   C   G   T   W   F   N   T   R   S   W   N   D   K   E


  5010 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa


!


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


!       R   Q   P   I   I   D   W   F   L   H   A   R   K   L   G


  5055 aga cag ccg att att gat tgg ttt cta cat gct cgt aaa tta gga


!


!      119 120 121 122 123 124 125 126 127 128 129 130 131 132 133


!       W   D   I   I   F   L   V   Q   D   L   S   I   V   D   K


  5100 tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa


!


!      134 135 136 137 138 139 140 141 142 143 144 145 146 147 148


!       Q   A   R   S   A   L   A   E   H   V   V   Y   C   R   R


  5145 cag gcg cgt tct gca tta gct gaa cat gtt gtt tat tgt cgt cgt


!


!      149 150 151 152 153 154 155 156 157 158 159 160 161 162 163


!       L   D   R   I   T   L   P   F   V   G   T   L   Y   S   L


  5190 ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt


!


!      164 165 166 167 168 169 170 171 172 173 174 175 176 177 178


!       I   T   G   S   K   M   P   L   P   K   L   H   V   G   V


  5235 att act ggc tcg 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


!       V   K   Y   G   D   S   Q   L   S   P   T   V   E   R   W


  5280 gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg


!


!      194 195 196 197 198 199 200 201 202 203 204 205 206 207 208


!       L   Y   T   G   K   N   L   Y   N   A   Y   D   T   K   Q


  5325 ctt tat act ggt aag aat ttg tat aac gca tat gat act aaa cag


!


!      209 210 211 212 213 214 215 216 217 218 219 220 221 222 223


!       A   F   S   S   N   Y   D   S   G   V   Y   S   Y   L   T


  5370 gct ttt tct agt aat tat gat tcc ggt gtt tat tct tat tta acg


!


!      224 225 226 227 228 229 230 231 232 233 234 235 236 237 238


!       P   Y   L   S   H   G   R   Y   F   K   P   L   N   L   G


  5415 cct tat tta tca cac ggt cgg tat ttc aaa cca tta aat tta ggt


!


!      239 240 241 242 243 244 245 246 247 248 249 250 251 252 253


!       Q   K   M   K   L   T   K   I   Y   L   K   K   F   S   R


  5460 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc


!


!      254 255 256 257 258 259 260 261 262 263 264 265 266 267 268


!       V   L   C   L   A   I   G   F   A   S   A   F   T   Y   S


  5505 gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt aca tat agt


!


!      269 270 271 272 273 274 275 276 277 278 279 280 281 282 283


!       Y   I   T   Q   P   K   P   E   V   K   K   V   V   S   Q


  5550 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag


!


!      284 285 286 287 288 289 290 291 292 293 294 295 296 297 298


!       T   Y   D   F   D   K   F   T   I   D   S   S   Q   R   L


  5595 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt


!


!      299 300 301 302 303 304 305 306 307 308 309 310 311 312 313


!       N   L   S   Y   R   Y   V   F   K   D   S   K   G   K   L


  5640 aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA


!                                                              PacI


!


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


!       I   N   S   D   D   L   Q   K   Q   G   Y   S   L   T   Y


  5685 ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat


!     PacI


!


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


!      i  I   D   L   C   T   V   S   I   K   K   G   N   S   N   E


                                                       (SEQ ID NO: 620)


!     iv                                                       M1  K


  5730   att gat tta tgt act gtt tcc att aaa aaa ggt aat tca aAT Gaa


!                                                             Start IV


!         344 345 346 347 348 349


!     i    I   V   K   C   N   .End of I


!     iv    L3  L   N5  V   I7  N    F  V10


  5775    att gtt aaa tgt aat TAA T TTT GTT


!  IV continued.....


  5800 ttc ttg atg ttt gtt tca tca tct tct ttt gct cag gta att gaa atg


  5848 aat aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca


  5896 ggc gaa tcc gtt att gtt tct ccc gat gta aaa ggt act gtt act gta


  5944 tat tca tct gac gtt aaa cct gaa aat cta cgc aat ttc ttt att tct


  5992 gtt tta cgt gct aat aat ttt gat atg gtt ggt tca att cct tcc ata


  6040 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca


  6088 tca tct gat aat cag gaa tat gat gat aat tcc gct cct tct ggt ggt


  6136 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat


  6184 aac gtt cgg gca aag gat tta ata cga gtt gtc gaa ttg ttt gta aag


  6232 tct aat act tct aaa tcc tca aat gta tta tct att gac ggc tct aat


  6280 cta tta gtt gtt TCT gca cct aaa gat att tta gat aac ctt cct caa


!                       ApaLI removed


  6328 ttc ctt tct act gtt gat ttg cca act gac cag ata ttg att gag ggt


  6376 ttg ata ttt gag gtt cag caa ggt gat gct tta gat ttt tca ttt gct


  6424 gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc


  6472 ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat


  6520 ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat


  6568 tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag aag


  6616 ggt tct atc tct gtT GGC CAg aat gtc cct ttt att act ggt cgt gtg


!                        MscI____


  6664 act ggt gaa tct gcc aat gta aat aat cca ttt cag acg att gag cgt


  6712 caa aat gta ggt att tcc atg agc gtt ttt cct gtt gca atg gct ggc


  6760 ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct


  6808 tct act cag gca agt gat gtt att act aat caa aga agt att gct aca


  6856 acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggc ctc act


  6904 gat tat aaa aac act tct caa gat tct ggc gta ccg ttc ctg tct aaa


  6952 atc cct tta atc ggc ctc ctg ttt agc tcc cgc tct gat tcc aac gag


  7000 gaa agc acg tta tac gtg ctc gtc aaa gca acc ata gta cgc gcc 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 gctttacggc acctcgaccc


  7240 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt


!                                  DraIII______


  7300 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac


!               DrdI__________


  7360 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga


  7420 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa


  7480 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga


!                                       PvuII.     BsmBI.


  7540 aaaaccaccc tGGATCC AAGCTT


!                  BamHI HindIII (1⁄2)


!                  Insert carrying bla gene


  7563    gcaggtg gcacttttcg gggaaatgtg cgcggaaccc


  7600 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct


!                                           BciVI


  7660 gataaatgct tcaataatat tgaaaaAGGA AGAgt


!                                  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 gct cac cca gaa acg ctg gtg aaa gta aaa gat gct


  7797 gaa gat cag ttg ggC gCA CGA Gtg ggt tac atc gaa ctg gat ctc aac agc


!                           BssSI...


!                       ApaLI removed


  7848 ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc


  7899 act ttt aaa gtt ctg cta tgt cat aca cta tta tcc cgt att gac gcc ggg


  7950 caa gaG CAA CTC GGT CGc cgg gcg cgg tat tct cag aat gac ttg gtt gAG


!            BcgI____________


ScaI


  8001 TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa


!      ScaI_


  8052 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta ctt


  8103 ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac atg


!               PvuI____


  8154 ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc


  8205 ata cca aac gac gag cgt gac acc acg atg cct gta gca atg cca aca acg


  8256 tTG CGC Aaa cta tta act ggc gaa cta ctt act cta gct tcc cgg caa caa


!       FspI....


!


  8307 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg


  8358 GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt


!       BglI_________


  8409 gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt


!       BsaI____


  8460 atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga aat


!                            AhdI___________


  8511 aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg TAA ctgt


!                                                              stop


  8560 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa


  8620 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt


  8680 cgttccactg tacgtaagac cccc


  8704 AAGCTT   GTCGAC tgaa tggcgaatgg cgctttgcct


!      HindIII  SalI..


!      (2/2)    HincII


  8740 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt


!


  8790 CCTGAGG


!      Bsu36I_


  8797      ccgat actgtcgtcg tcccctcaaa ctggcagatg


  8832 cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg


  8892 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc


  8952 tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg


  9012 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaATTTAAA


!


SwaI...


  9072 Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat


!                                                             RBS?


  9131 ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt tgc


!      Start gene II


  9182 tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa ata


!                                                    BglII...


  9233 gct acc ctc tcc ggc atg aat tta tca gct aga acg 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 tca ggc att gca ttt aaa ata tat gag ggt tct aaa aat ttt


  9386 tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt cat


  9437 aat gtt ttt ggt aca acc gat tta gct tta tgc tct gag gct tta ttg ctt


  9488 aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt !


9532


! gene II continues
















TABLE 21B





Sequence of MALIA3, condensed LOCUS MALIA3 9532 


CIRCULAR ORIGIN (SEQ ID NO: 451)
















1
AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT


 


61
ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT


 


121
CGTTCGCAGA ATTGGGAATC AACTGTTACA TGGAATGAAA CTTCCAGACA CCGTACTTTA


 


181
GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA


 


241
TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG


 


301
TTGGAGTTTG CTTCCGGTCT GGTTCGCTTT GAAGCTCGAA TTAAAACGCG ATATTTGAAG


361
TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT


 


421
CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA


 


481
TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT


 


541
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


 


1681
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


 


7621
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


 


8461
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


 


8821
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








HuCμ-FOR (1st PCR)
5'-TGG AAG AGG CAC GTT CTT TTC TTT-3' (SEQ ID NO: 457)


HuCμ-Nested (2nd PCR)
5' CTT TTC TTT GTT GCC GTT GGG GTG-3' (SEQ ID NO: 458)


 







Kappa light chain








HuCkFor (1st PCR)
5'-ACA CTC TCC CCT GTT GAA GCT CTT-3' (SEQ ID NO: 459)


HuCkForAscl(2nd PCR)
5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA ACA CTC TCC



CCT GTT GAA GCT CTT-3'               (SEQ ID NO: 460)


 







Lambda light chain HuClambdaFor (1st PCR)








HuCL2-FOR
5'-TGA ACA TTC TGT AGG GGC CAC TG-3' (SEQ ID NO: 461)


HuCL7-FOR
5'-AGA GCA TTC TGC AGG GGC CAC TG-3' (SEQ ID NO: 462)


 







HuClambdaForAscl (2nd PCR)








HuCL2-FOR-ASC
5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA TGA ACA TTC



TGT AGG GGC CAC TG-3' (SEQ ID NO: 463)


HuCL7-FOR-ASC
5'-ACC GCC TCC ACC GGG CGC GCC TTA TTA AGA GCA TTC



TGC AGG GGC CAC TG-3' (SEQ ID NO: 464)


 







GeneRAcer 5' Primers provided with the kit (Invitrogen)








5'A 1st PCR
(SEQ ID NO: 465) 5'CGACTGGAGCACGAGGACACTGA 3'


5'NA 2nd pCR
5'GGACACTGACATGGACTGAAGGAGTA-3' (SEQ ID NO: 466)
















TABLE 23





ONs used in Capture of kappa light chains using CJ method and BsmAI


All ONs are written 5' to 3'.















REdapters (6)








ON_20SK15012
                         gggAggATggAgAcTgggTc (SEQ ID NO: 467)


ON_20SK15L12
                         gggAAgATggAgAcTgggTc (SEQ ID NO: 468)


ON_20SK15A17
                         gggAgAgTggAgAcTgAgTc (SEQ ID NO: 469)


ON_20SK15A27
                         gggTgccTggAgAcTgcgTc (SEQ ID NO: 470)


ON_20SK15A11
                         gggTggcTggAgAcTgcgTc (SEQ ID NO: 471)


ON_20SK15B3
                         gggAgTcTggAgAcTgggTc (residues 1-20 of SEQ ID NO: 477)


 







Bridges (6)








kapbri1012
gggAggATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 472)


kapbri1L12
gggAAgATggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 473)


kapbri1Al7
gggAgAgTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 474)


kapbri1A27
gggTgccTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 475)


kapbri1All
gggTggcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 476)


kapbri1B3
gggAgTcTggAgAcTgggTcATcTggATgTcTTgTgcAcTgTgAcAgAgg (SEQ ID NO: 477)


 







Extender (5' biotinylated)








kapext1bio
ccTcTgTcAcAgTgcAcAAgAcATccAgATgAcccAgTcTcc (SEQ ID NO: 478)


 







Primers








kaPCRt1
ccTcTgTcAcAgTgcAcAAgAc (SEQ ID NO: 479)


kapfor
5'-aca ctc tcc cct gtt gaa get ctt-3' (SEQ ID NO: 480)
















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



10x turbo PCR buffer
1x



turbo Pfu
4U



dNTPs
200 μM each



kaPCRt1
300 nM



kapfor
300 nM

















TABLE 25





h3401-h2 captured Via CJ with BsmAI


(Nucleotide sequence is SEQ ID NO: 481,


Amino acid sequence is SEQ ID NO: 482)















! 1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


! S   A   Q   D   I   Q   M   T   Q  S   P   A   T   L   S


 aGT GCA Caa gac atc cag atg acc cag tct cca gcc acc ctg tct


 


! ApaLI... a gcc acc ! L25,L6,L20,L2,L16,A11


! Extender................................ Bridge...


 


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


! V   S   P   G   E   R   A   T   L   S   C   R   A   S   Q


  gtg tct cca ggg gaa agg gcc acc ctc tcc tgc agg gcc agt cag


 


! 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


! S   V   S   N   N   L   A   W   Y   Q   Q   K   P   G   Q


  agt gtt agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag


 


! 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


! V   P   R   L   L   I   Y   G   A   S   T   R   A   T   D


  gtt ccc agg ctc ctc atc tat ggt gca tcc acc agg gcc act gat


 


! 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


! I   P   A   R   F   S   G   S   G   S   G   T   D   F   T


  atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act


 


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


! L   T   I   S   R   L   E   P   E   D   F   A   V   Y   Y


  ctc acc atc agc 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


! C   Q   R   Y   G   S   S   P   G    W   T   F   G   Q   G


  tgt cag cgg tat ggt agc tca ccg ggg tgg acg ttc ggc caa ggg


 


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


!  T   K   V   E   I   K   R   T   V   A   A   P   S   V   F


  acc aag gtg gaa atc aaa cga act gtg get gca cca tct gtc ttc


 


! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!  I   F   P   P   S   D   E   Q   L   K   S   T   A   A   S


  atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct


 


! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!  V   V   C   L   L   N   N   F   Y   P   R   E   A   K   V


  gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta


 


! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!  Q   W   K   V   D   N   A   L   Q   S   G   N   S   Q   E


  cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag


 


! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!  S   V   T   E   Q   D   S   K   D   S   T   Y   S   L   S


  agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc


 


! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!  S   T   L   T   L   S   K   A   D   Y   E   K   H   K   V


  agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc


 


! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!  Y   A   C   E   V   T   H   Q   G   L   S   S   P   V   T


  tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cct gtc aca


 


! 211 212 213 214 215 216 217 218 219 220 221 222 223


!  K   S   F   N   K   G   E   C   K   G   E   F  A


  aag agc ttc aac aaa gga gag tgt aag ggc gaa ttc gc
















TABLE 26





h3401-d8 KAPPA captured with CJ and BsmAI


(Nucleotide sequence is SEQ ID NO: 484;


Amino acid sequence is SEQ ID NO: 485)















! 1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


! S   A   Q   D   I   Q   M   T   Q  S   P   A   T   L   S


 aGT GCA Caa gac atc cag atg acc cag tct cct gcc acc ctg tct


 


!ApaLI...Extender...........a gcc acc ! L25,L6,L20,L2,L16,A11


!A GCC ACC CTG TCT ! L2 (SEQ ID NO: 483)


 


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


! V   S   P   G   E   R   A   T   L   S   C   R   A   S   Q


  gtg tct cca ggt gaa aga gcc acc ctc tcc tgc agg gcc 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


  N   L   L   S   N   L   A   W   Y   Q   Q   K   P   G   Q


  aat ctt ctc agc aac tta gcc tgg tac cag cag aaa cct ggc cag


 


! 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


! A   P   R   L   L   I   Y   G   A   S   T   G   A   I   G


  gct ccc agg ctc ctc atc tat ggt gct tcc acc ggg gcc att ggt


 


! 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


! I   P   A   R   F   S   G   S   G   S   G   T   E   F   T


  atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gag ttc act


 


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


! L   T   I   S   S   L   Q   S   E   D   F   A   V   Y   F


  ctc acc atc agc agc ctg cag tct gaa gat ttt gca gtg tat ttc


 


! 91  92  93  94  95  96  97  98  99  100 101 102 103 104 105


! C   Q   Q   Y   G   T   S   P   P    T   F   G   G   G   T


  tgt cag cag tat ggt acc tca ccg ccc act ttc ggc gga ggg acc


 


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


!  K   V   E   I   K   R   T   V   A   A   P   S   V   F   I


  aag gtg gag atc aaa cga act gtg get gca cca tct gtc ttc atc


 


! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


   F   P   P   S   D   E   Q   L   K   S   G   T   A   S   V


  ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt


 


! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!  V   C   P   L   N   N   F   Y   P   R   E   A   K   V   Q


  gtg tgc ccg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag


 


! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!  W   K   V   D   N   A   L   Q   S   G   N   S   Q   E   S


  tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt


 


! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!  V   T   E   Q   D   N   K   D   S   T   Y   S   L   S   S


  gtc aca gag cag gac aac aag gac agc acc tac agc ctc agc agc


 


! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!  T   L   T   L   S   K   V   D   Y   E   K   H   E   V   Y


  acc ctg acg ctg agc 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


!  A   C   E   V   T   H   Q   G   L   S   S   P   V   T   K


  gcc tgc gaa gtc acc cat cag ggc ctt agc tcg ccc gtc acg aag


 


! 211 212 213 214 215 216 217 218 219 220 221 222 223


!  S   F   N   R   G   E   C   K   K   E   F   V


  agc ttc aac agg gga gag tgt aag aaa gaa ttc gtt t
















TABLE 27





V3-23 VH framework with variegated codons shown















!(Nucleotide sequence is SEQ ID NO: 486; Amino acid sequence is SEQ ID NO: 487)


!                    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 cgg gtc ggc cgg tac cgg


!          Scab.......SfiI........


!                        NgoMI....


!                           NcoI....


 








!  !  ! ! !
                    





 













 


 





 


 





 


 





 


 





 








 








 








 


            





 








 








 


!


!CDR1 diversity


!


 








 


!<1> stands for an equimolar mix of {ADEFGHIKLMNPQRSTVWY}; no C


!                                                 (this is no a sequence)


!


!CDR2 diversity


 








 


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


!<2> is an equimolar mixture of {YRWVGS}; no ACDEFHIKLMNPQT


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
















TABLE 28





Stuffer used in VH (SEQ ID NO: 498)
















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 AAACATTAAC 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 CDR1-2 and CDR3 2000.12.13


!


! Ngene = 6680


! Useful REs (cut MAnoLI fewer than 3 times) 2000.06.05


!


! Non-cutters









!Acc65I Ggtacc
AfeI AGCgct
AvrII Cctagg


!BsaBI GATNNnnatc
BsiWI Cgtacg 
BsmFI Nnnnnnnnnnnnnnngtccc


(SEQ ID NO: 499)

      (SEQ ID NO: 500)








!BsrGI Tgtaca
BstAPI GCANNNNntgc BstBI Ttcgaa



      (SEQ ID NO: 501)









!BstZ17I GTAtac
BtrI CACgtg
Ecl136I GAGctc


!EcoRV GATatc
FseI GGCCGGcc
KpnI GGTACc


!MscI TGGcca
NruI TCGcga
NsiI ATGCAt


!PacI TTAATtaa
PmeI GTTTaaac
PmlI CACgtg


!PpuMI RGgwccy
PshAI GACNNnngtc 
SacI GAGCTc



 (SEQ ID NO: 502)



!SacII CCGCgg
SbfI CCTGCAgg
SexAI Accwggt


!Sgfl GCGATcgc
SnaBI TACgta
SpeI Actagt


!SphI GCATGc
Sse8387I CCTGCAgg
StuI AGGcct








!SwaI ATTTaaat
XmaI Cccggg







!


! cutters








! Enzymes that cut more than
3 times.








!AlwNI CAGNNNctg
5








!BsgI ctgcac
4


!BsrFI Rccggy
 5


!EarI CTCTTCNnnn
   4








(SEQ ID NO: 625)



!FauI nNNNNNNGCGGG
 10


! (SEQ ID NO: 503)



!Enzymes that cut from 1 to
3 times.







!








!EcoO109I RGgnccy
       3     7 2636 4208


!BssSI Ctcgtg
  1    12


!-′′ - Cacgag
 1   1703


!BspHI Tcatga
   3   43  148  1156


!AatII GACGTc
    1   65


!BciVI GTATCCNNNNNN
           2    140  1667


(SEQ ID NO: 504)



!Eco57I CTGAAG
      1  301


!-′′ - cttcag
2  1349


!Aval Cycgrg
  3   319  2347  6137


!BsiHKAI GWGCWc
        3   401  2321  4245


!HgiAI GWGCWc
      3   401  2321  4245


!BcgI gcannnnnntcg
     1  461


(SEQ ID NO: 505)



!SacI AGTact
  1  505


!PvuI CGATcg
   3  616  3598  5926


!FspI TGCgca
  2  763  5946


!BglT GCCNNNNnggc
      3   864  2771  5952


(SEQ ID NO: 506)



!BpmI CTGGAG
    1    898


!-′′ - ctccag
1 4413


!BsaI GGTCTCNnnnn
      1    916


(SEQ ID NO: 507)



!AhdI GACNNNnngtc
      1    983


(SEQ ID NO: 508)



!Eam1105I GACNNNnngtc
         1   983


(SEQ ID NO: 509)



!DrdI GACNNNNnngtc
      3    1768  6197  6579


(SEQ ID NO: 510)



!SapI gaagagc
 1  1998


!PvuII CAGctg
  3  2054  3689  5896


!PflMI CCANNNNntgg
       3  2233  3943  3991


(SEQ ID NO: 511)



!HindIII Aagctt
  1  2235


!ApaLI Gtgcac
   1  2321


!BspMI Nnnnnnnnngcaggt
        1  2328


(SEQ ID NO: 512)



!-′′ - ACCTGCNNNNn
        2  3460


(SEQ ID NO: 513)



!PstI CTGCAg
   1  2335


!AccI GTmkac
   2  2341  2611


!HincII GTYrac
   2  2341  3730


!SalI Gtcgac
 1  2341


!TliI Ctcgag
 1  2347


!XhoI Ctcgag
  1  2347


!BbsI gtcttc
2  2383  4219


!BlpI GCtnagc
 1  2580 


!EspI GCtnagc
 1  2580


!SgrAI CRccggyg
  1  2648


!AgeI Accggt
 2  2649  4302


!AscI GGcgcgcc
   1  2689


!BssHII Gcgcgc
  1  2690


!SfiI GGCCNNNNaggcc
       1  2770


(SEQ ID NO: 514)



!NaeI GCCggc
 2  2776  6349


!NgoMIV Gccggc
  2  2776  6349


!BtgI Ccrygg
3  2781  3553  5712


!DsaI Ccrygg
3  2781  3553  5712


!NcoI Ccatgg
 1  2781


!StyI Ccwwgg
 3  2781  4205  4472


!MfeI Caattg
1  2795


!BspEI Tccgga
  1  2861


!BglII Agatct
1  2872


!BclI Tgatca
1  2956


!Bsu36I CCtnagg
   3  3004  4143  4373


!XcmI CCANNNNNnnnntgg
         1  3215


(SEQ ID NO: 515)



!MluI Acgcgt
   1  3527


!HpaI GTTaac
    1  3730


!XbaI Tctaga
   1  3767


!



!AflII Cttaag
  1  3811


!BsmI NGcattc
   1  3821


!-′′ - GAATGCN
    1  4695


!RsrII CGgwccg
   1  3827


!NheI Gctagc
  1  4166


!BstEII Ggtnacc
   1  4182


!BsmBI CGTCTCNnnnn
       2  4188  6625


(SEQ ID NO: 516)



!-′′ - Nnnnnngagacg
   1  6673


(SEQ ID NO: 517)



!ApaI GGGCCc
   1  4209


!BanII GRGCYc
   3  4209  4492  6319


!Bsp1201 Gggccc
  1  4209


!PspOMI Gggccc
   1  4209


!BseRI NNnnnnnnnnctcctc
      1  4226


(SEQ ID NO: 518)



!-′′ - GAGGAGNNNNNNNNNN
           1  4957


(SEQ ID NO: 519)



!EcoNI CCTNNnnnagg
      1  4278


(SEQ ID NO: 520)



!PflFI GACNnngtc
    1  4308


!Tth111I GACNnngtc
     1  4308


!KasI Ggcgcc
2  4327  5967


!BstXI CCANNNNNntgg
      1  4415


(SEQ ID NO: 521)



!NotI GCggccgc
 1  4507


!EagI Cggccg
1  4508


!BamHI Ggatcc
 1  5169


!BspDI ATcgat
 1  5476


!NdeI CAtatg
1  5672


!EcoRI Gaattc
1  5806


!PsiI TTAtaa
1  6118


!DraIII CACNNNgtg
    1  6243


!BsaAI YACgtr
 1  6246







!-------------------------------------------


(Nucleotide sequence is SEQ ID NO: 522 and Amino acid sequence is


SEQ ID NO: 523, respectively)


   1   gacgaaaggg cCTCGTGata cgcctatttt tataggttaa tgtcatgata ataatggttt


!            BssSI.(1/2)


  61   cttaGACGTC aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt


!       AatI.


 121   tctaaataca ttcaaatatG TATCCgctca tgagacaata accctgataa atgcttcaat


!                 BciVI..(1 of 2)


 181   aatattgaaa aaggaagagt


! Base # 201 to 1061 = ApR gene from pUC119 with some RE sites removed


!


!        1   2   3   4   5   6   7   8   9   10  11  12  13  14  15


!        fM  S   I   Q   H   F   R   V   A   L   I   P   F   F   A


 201    atg agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg


!


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


!        A   F   C   L   P   V   F   A   H   P   E   T   L   V   K


 246    gca ttt tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa


!


!        31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!        V   K   D   A   E   D   Q   L   G   A   R   V   G   Y   I


 291    gta aaa gat gct gaa gat cag ttg ggt gcc cga gtg ggt tac atc


!


!        46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!        E   L   D   L   N   S   G   K   I   L   E   S   F   R   P


 336    gaa ctg gat ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc


!


!        61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!        E   E   R   F   P   M   M   S   T   F   K   V   L   L   C


 381    gaa gaa cgt ttt cca atg atg agc act ttt aaa gtt ctg cta tgt


!


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


!        G   A   V   L   S   R   I   D   A   G   Q   E   Q   L   G


 426    ggc gcg gta tta tcc cgt att gac gcc ggg caa gaG CAa ctc ggT


!                                    BcgI.........


!


!        91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!        R   R   I   H   Y   S   Q   N   D   L   V   E   Y   S   P


 471    CGc cgc ata cac tat tct cag aat gac ttg gtt gAG TAC Tca cca


!..BcgI....                           ScaI....


!


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


!        V   T   E   K   H   L   T   D   G   M   T   V   R   E   L


 516    gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga gaa tta


!


!       121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!        C   S   A   A   I   T   M   S   D   N   T   A   A   N   L


 561    tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta


!


!       136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!        L   L   T   T   I   G   G   P   K   E   L   T   A   F   L


 606    ctt ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg


!               PvuI.... (1/2)


!


!       151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!        H   N   M   G   D   H   V   T   R   L   D   R   W   E   P


 651    cac aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg


!


!       166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!        E   L   N   E   A   I   P   N   D   E   R   D   T   T   M


 696    gag ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg 


!


!       181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!        P   V   A   M   A   T   T   L   R   K   L   L   T   G   E


 741    cct gta GCA ATG gca aca acg tTG CGC Aaa cta tta act ggc gaa


!           BsrDI..(1/2)  FspI.... (1/2)


!


!       196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!        L   L   T   L   A   S   R   Q   Q   L   I   D   W   M   E


 786    cta ctt act cta gct tcc cgg caa caa tta ata gac tgg atg gag


!


!       211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!        A   D   K   V   A   G   P   L   L   R   S   A   L   P   A


 831    gcg gat aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct


!


!       226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!        G   W   F   I   A   D   K   S   G   A   G   E   R   G   S


 876    ggc tgg ttt att gct gat aaa tCT GGA Gcc ggt gag cgt gGG TCT


!                          BpmI....(1/2)    BsaI....


!


!       241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!        R   G   I   I   A   A   L   G   P   D   G   K   P   S   R


 921    Cgc ggt atC ATT GCa gca ctg ggg cca gat ggt aag ccc tcc cgt


!BsaI......   BsrDI...(2/2)


!


!       256 257 258 259 260 261 262 263 264 265 266 267 268 269 270


!        I   V   V   I   Y   T   T   G   S   Q   A   T   M   D   E


 966    atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa


!                     AhdI........


!


!       271 272 273 274 275 276 277 278 279 280 281 282 283 284 285


!        R   N   R   Q   I   A   E   I   G   A   S   L   I   K   H


 1011   cga cct aga cag atc gct gag ata ggt gcc tca ctg agg aag cat


!


!       286 287


!        W


 1056   tgg taa


 1062                                  ctgtcagac caagtttact


 1081   catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga


 1141   tcctttttga taatctcatg accaaaatcc cttaacgtga gttttctggc cactgagcgt


 1201   cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct


 1261   gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc


 1321   taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc


 1381   ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc


 1441   tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg


 1501   ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt


 1561   cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg


 1621   agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacagGTAT CCggtaagcg


!                                   BciVI.. (2 of 2)


 1681   gcagggtcgg aacaggagag cgCACGAGgg agcttccagg gggaaacgcc tggtatgttt


!                  BssSI.(2/2)


 1741   atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag


 1801   gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt


 1861   gctggccttt tgctcACATG Ttctttcctg cgttatcccc tgattctgtg gataaccgta


!              PciI...


 1921   ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt


 1981   cagtgagcga ggaagcgGAA GAGCgcccaa tacgcaaacc gcctctcccc gcgcgttggc


!               SapI....


 2041   cgattcatta atgCAGCTGg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca


!              PvuII.(1/3)


 2101   acgcaaTAA TGTgagttag ctcactcatt aggcaccccca ggcTTTACAc tttatgcttc


!        ..35..      Plac            ..-10.


 2161   cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacaCAGGA AACAGCTATG


!                                    M13Rev_seq_primer


 2221   ACcatgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac


!            PflMI.......


!             Hind3.


!signal::linker::CLight


!


        (Amino acid sequence is SEQ ID NO: 524)


!        1   2   3   4   5   6   7   8   9   10  11  12  13  14  15


!        fM  K   K   L   L   F   A   I   P   L   V   V   P   F   Y 


 2269   gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat


!


!                   Linker....................End of FR4


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


!        S   H   S   A   Q   V   Q   L   Q   V   D   L   E   I   K


 2314   tct cac aGT GCA Cag gtc caa CTG CAG GTC GAC CTC GAG atc aaa


!           ApaLI.....  PstI...     XhoI...


!                      BspMI...


!                         SalI...


!                         AccI...(1/2)


!                         HincII.(1/2)


!


!Vlight domains could be cloned in as ApaLI-XhoI fragments.


!VL-CL(kappa) segments can be cloned in as ApaLI-AscI fragments. <------


!


!


!        31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!        R   G   T   V   A   A   P   S   V   F   I   F   P   P   S


 2359   cgt gga act gtg gct gca cca tct GTC TTC atc ttc ccg cca tct


!                          BbsI...(1/2)


!


!        46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!        D   E   Q   L   K   S   G   T   A   S   V   V   C   L   L


 2404   gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg


!


!        61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!        N   N   F   Y   P   R   E   A   K   V   Q   W   K   V   D


 2449   aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat


!


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


!        N   A   L   Q   S   G   N   S   Q   E   S   V   T   E   Q


 2494   aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag


!


!        91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!        D   S   K   D   S   T   Y   S   L   S   S  T   L   T   L


 2539   gac agc aag gac agc acc tac agc ctc agc agc acc ctg acG CTG


!                                      EspI...


!


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


!        S   K   A   D   Y   E   K   H   K   V   Y   A   C   E   V


 2584   AGC aaa gca gac tac gag aaa cac aaa GTC TAC gcc tgc gaa gtc


! ...EspI....                AccI...(2/2)


!


!       121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!        T   H   Q   G   L   S   S   P   V   T   K   S   F   N   R


 2629   acc cat cag ggc ctg agt tcA CCG GTg aca aag agc ttc aag agg


!                     AgeI....(1/2)


!


!       136 137 138 139 140


!        G   E   C   .   .


 2674   gga gag tgt taa taa GG CGCGCCaatt


!                   AscI.....


!                   BssHII.


!


 2701   ctatttcaag gagacagtca ta


!


!PelB::3-23(stuffed)::CH1::III fusion gene


!


        (Amino acid sequence is SEQ ID NO: 525)


!         1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!        M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L


 2723   atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc


!


!-------------------------------------


!


!        16  17  18  19  20  21  22


!        A   A   Q   P   A   M   A


 2768   gcG GCC cag ccG GCC atg gcc


!        SfiI..........


!            NgoMIV..(1/2)


!               NcoI....


!








!








!








!


!       Stuffer for CDR1, FR2, and CDR2------------------------------->


!       There are no stop codons in this stuffer.


 2867                                  gcttcAGATC Tgtttgcctt 


!                                      BglII..


 2887  tttgtggggt ggtgcagatc gcgttacgga gatcgaccga ctgcttgagc aaaagccacg


 2947  cttaactgcT GATCAggcat gggatgttat tcgccaaacc agtcgtcagg atcttaacct


!          BcII...


 3007  gaggcttttt ttacctactc tgcaagcagc gacatctggt ttgacacaga gcgatccgcg


 3067  tcgtcagttg gtagaaacat taacacgttg ggatggcatc aatttgctta atgatgatgg


 3127  taaaacctgg cagcagccag gctctgccat cctgaacgtt tggctgacca gtatgttgaa


 3187  gcgtaccgta gtggctgccg tacctatgCC Atttgataag TGGtacagcg ccagtggcta


!                      XcmI..........


 3247  cgaaacaacc caggacggcc caactggttc gctgaatata agtgttggag caaaaatttt


 3307  gtatgaggcg gtgcagggag acaaatcacc aatcccacag gcggttgatc tgtttgctgg


 3367  gaaaccacag caggaggttg tgttggctgc gctggaagat acctgggaga ctctttccaa


 3427  acgctatggc aataatgtga gtaactggaa aacacctgca atggccttaa cgttccgggc


 3487  aaataatttc tttggtgtac cgcaggccgc agcggaagaa ACGCGTcatc aggcggagta


!                                MluI..


 3547  tcaaaaccgt ggaacagaaa acgatatgat tgttttctca ccaacgacaa gcgatcgtcc


 3607  tgtgcttgcc tgggatgtgg tcgcacccgg tcagagtggg tttattgctc ccgatggaac


 3667  agttgataag cactatgaag atcagctgaa aatgtacgaa aattttggcc gtaagtcgct


!                   PvuII.


 3727  ctgGTTAACg aagcaggatg tggaggcgca taaggagtcg


!      HpaI..


!      HincII(2/2)


!








!








!


!       q h  s   p  nt-.


 3834  gg caa cat tct 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 ctattgcctt ttgaaatgaa ccctaaggtg tataaccccc ag


 4164      aa GCTAGC ctgcggcttc


!          NheI..


!








!


       (Amino acid sequence is SEQ ID NO: 527)


!      136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!       A   S   T   K   G   P   S   V   F   P   L   A   P   S   S


 4198  gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc


!


!      151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!       K   S   T   S   G   G   T   A   A   L   G   C   L   V   K


 4243  aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag


!


!      166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!       D   Y   F   P   E   P   V   T   V   S   W   N   S   G   A


 4288  gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc


!


!      181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!       L   T   S   G   V   H   T   F   P   A   V   L   Q   S   S


 4333  ctg acc agc ggc gtc cac acc ttc ccg get gtc cta cag tcc tca


!


!      196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!       G   L   Y   S   L   S   S   V   V   T   V   P   S   S   S


 4378  gga Ctc tac tcc ctc agc agc gta gtg acc gtg ccc tcc agc agc


!


!      211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!       L   G   T   Q   T   Y   I   C   N   V   N   H   K   P   S


 4423  ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc


!


!      226 227 228 229 230 231 232 233 234 235 236 237 238


!       N   T   K   V   D   K   K   V   E   P   K   S   C


 4468  aac acc aag gtg 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


!       A   A   A   H   H   H   H   H   H   G   A   A


 4507  GCG GCC GCa cat cat cat cac cat cac ggg gcc gca


!      NotI.....


!      EagI....


!


!      151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!       E   Q   K   L   I   S   E   E   D   L   N   G   A   A   .


 4543  gaa caa aaa etc atc tca gaa gag gat ctg aat ggg gcc gca tag


!


!      Mature III --------------------------------->...


!      166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!       T   V   E   S   C   L   A   K   P   H   T   E   N   S   F


 4588  act gtt gaa agt tgt tta gca aaa cct cat aca gaa aat tca ttt


!


!      181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!       T   N   V   W   K   D   D   K   T   L   D   R   Y   A   N


 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


!       Y   E   G   C   L   W   N   A   T   G   V   V   V   C   T


 4678  tat gag ggc tgt ctg tgG AAT GCt aca ggc gtt gtg gtt tgt act


!                  BsmI....


!


!      211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!       G   D   E   T   Q   C   Y   G   T   W   V   P   I   G   L


 4723  ggt gac gaa act cag tgt tac ggt aca tgg gtt cet att ggg ctt


!


!      226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!       A   I   P   E   N   E   G   G   G   S   E   G   G   G   S


 4768  gct atc cct gaa aat gag ggt ggt ggc tct gag ggt ggc ggt tct


!


!      241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!       E   G   G   G   S   E   G   G   G   T   K   P   P   E   Y


 4813  gag ggt ggc ggt tct gag ggt ggc ggt act aaa cct cct gag tac


!


!      256 257 258 259 260 261 262 263 264 265 266 267 268 269 270


!       G   D   T   P   I   P   G   Y   T   Y   I   N   P   L   D


 4858  ggt gat aca cct att ccg ggc tat act tat atc aac cct ctc gac


!


!      271 272 273 274 275 276 277 278 279 280 281 282 283 284 285


!       G   T   Y   P   P   G   T   E   Q   N   P   A   N   P   N


 4903  ggc act tat ccg cct ggt act gag caa aac ccc gct aat cct aat


!


!      286 287 288 289 290 291 292 293 294 295 296 297 298 299 300


!       P   S   L   E   E   S   Q   P   L   N   T   F   M   F   Q


 4948  cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag


!            BseRI..(2/2)


!


!      301 302 303 304 305 306 307 308 309 310 311 312 313 314 315


!       N   N   R   F   R   N   R   Q   G   A   L   T   V   Y   T


 4993  aat aat agg ttc cga aat agg cag ggt gca tta act gtt tat acg


!


!      316 317 318 319 320 321 322 323 324 325 326 327 328 329 330


!       G   T   V   T   Q   G   T   D   P   V   K   T   Y   Y   Q


 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


!       Y   T   P   V   S   S   K   A   M   Y   D   A   Y   W   N


 5083  tac act cct gta tca tca aaa gcc atg tat gac gct tac tgg aac


!


!      346 347 348 349 350 351 352 353 354 355 356 357 358 359 360


!       G   K   F   R   D   C   A   F   H   S   G   F   N   E   D


 5128  ggt aaa ttc aga gac tgc gct ttc cat tct ggc ttt aat gaG GAT


!                                        BamHI..


!


!      361 362 363 364 365 366 367 368 369 370 371 372 373 374 375


!       P   F   V   C   E   Y   Q   G   Q   S   S   D   L   P   Q


 5173  CCa ttc gtt tgt gaa tat caa ggc caa tcg tct gAC CTG Cct caa


!BamHI...                              BspMI...(2/2)


!


!      376 377 378 379 380 381 382 383 384 385 386 387 388 389 390


!       P   P   V   N   A   G   G   G   S   G   G   G   S   G   G


 5218  cct cct gtc aat gct ggc ggc ggc tct ggt ggt ggt tct ggt ggc


!


!      391 392 393 394 395 396 397 398 399 400 401 402 403 404 405


!       G   S   E   G   G   G   S   E   G   G   G   S   E   G   G


 5263  ggc tct gag ggt ggc ggc tct gag ggt ggc ggt tct gag ggt ggc


!


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


!       G   S   E   G   G   G   S   G   G   G   S   G   S   G   D


 5308  ggc tct gag ggt ggc ggt tcc ggt ggc ggc tcc ggt tcc ggt gat


!


!      421 422 423 424 425 426 427 428 429 430 431 432 433 434 435


!       F   D   Y   E   K   M   A   N   A   N   K   G   A   M   T


 5353  ttt gat tat gaa aaa atg gca aac get aat aag ggg get atg acc


!


!      436 437 438 439 440 441 442 443 444 445 446 447 448 449 450


!       E   N   A   D   E   N   A   L   Q   S   D   A   K   G   K


 5398  gaa aat gcc gat gaa aac gcg cta cag tct gac get aaa ggc aaa


!


!      451 452 453 454 455 456 457 458 459 460 461 462 463 464 465


!       L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F


 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


!       I   G   D   V   S   G   L   A   N   G   N   G   A   T   G


 5488  att ggt gac gtt tcc ggc ctt get aat ggt aat ggt get act ggt


!


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


!       D   F   A   G   S   N   S   Q   M   A   Q   V   G   D   G


 5533  gat ttt get ggc tct aat tcc caa atg get caa gtc ggt gac ggt


!


!      496 497 498 499 500 501 502 503 504 505 506 507 508 509 510


!       D   N   S   P   L   M   N   N   F   R   Q   Y   L   P   S


 5578  gat aat tca cct tta atg aat aat ttc cgt caa tat tta cct tct


!


!      511 512 513 514 515 516 517 518 519 520 521 522 523 524 525


!       L   P   Q   S   V   E   C   R   P   Y   V   F   G   A   G


 5623  ttg cct cag tcg gtt gaa tgt cgc cct tat gtc ttt ggc get ggt


!


!      526 527 528 529 530 531 532 533 534 535 536 537 538 539 540


!       K   P   Y   E   F   S   I   D   C   D   K   I   N   L   F


 5668  aaa cCA TAT Gaa ttt tct att gat tgt gac aaa ata aac tta ttc


!      NdeI....


!


!      541 542 543 544 545 546 547 548 549 550 551 552 553 554 555


!       R   G   V   F   A   F   L   L   Y   V   A   T   F   M   Y


 5713  cgt ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat


!


!      556 557 558 559 560 561 562 563 564 565 566 567 568 569 570


!       V   F   S   T   F   A   N   I   L   R   N   K   E   S   .


 5758  gta ttt tcg acg 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


!                                         PvuI..


 5931  Gcccttccca acagtTGCGC Agcctgaatg gcgaatGGCG CCtgatgcgg tattttctcc


!...PvuI... (3/3) FspI... (2/2) KasI...(2/2)


 5991  ttacgcatct gtgcggtatt tcacaccgca tataaattgt aaacgttaat attttgttaa


 6051  aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca


 6111  aaatcccTTA TAAatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga


!        PsiI...


 6171  acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc


 6231  agggcgatgg ccCACtacGT Gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc


!           DralTI....


 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  agggcgcgta ctatggttgc tttgacgggt gcagtctcag tacaatctgc tctgatgccg


 6531  catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc


 6591  tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga


 6651  ggttttcacc gtcatcaccg aaacgcgcga
















TABLE 30





Oligonucleotides used to clone CDR1/2 diversity















All sequences are 5′ to 3′.


 


1) ON_CD1Bsp, 30 bases (SEQ ID NO: 528)


 


A c c T c A c T g g  c  T  T  c  c  g  g  A


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18


 


T  T  c  A  c  T  T  T  c  T  c  T


19 20 21 22 23 24 25 26 27 28 29 30


 


2) ON_Br12, 42 bases (SEQ ID NO: 529)


 


A g A A A c c c A c  T  c  c  A  A  A  c  c


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18


 


T  T  T  A  c  c  A  g  g  A  g  c  T  T  g  g  c


19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35


 


g  A  A  c  c  c  A


36 37 38 39 40 41 42


 


3) ON_CD2Xba, 51 bases (SEQ ID NO: 530)


 


g g A A g g c A g T  g  A  T  c  T  A  g  A


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18


 


g  A  T  A  g  T  g  A  A  g  c  g  A  c  c  T  T


19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35


 


T  A  A  c  g  g  A  g  T  c  A  g  c  A  T  A


36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51


 


4) ON_BotXba, 23 bases (SEQ ID NO: 531)


 


g g A A g g c A g T  g  A  T  c  T  A  g  A


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


(SEQ ID NOS 532-546,respectively in order of appearance)

















ON_Lam1aB7(rc)   
........................GTGCTGACTCAGCCACCCTC.
20


ON_Lam2aB7(rc)
........................GCCCTGACTCAGCCTGCCTC.
20


ON_Lam31B7(rc)
........................GAGCTGACTCAGG.ACCCTGC
20


ON_Lam3rB7(rc)
........................GAGCTGACTCAGCCACCCTC.
20


ON_LamHf1cBrg(rc)
CCTCGACAGCGAAGTGCACAGAGCGTCTTGACTCAGCC.......
38


ON_LamHf1cExt
CCTCGACAGCGAAGTGCACAGAGCGTCTTG...............
30


ON_LamHf2b2Brg(rc) 
CCTCGACAGCGAAGTGCACAGAGCGCTTTGACTCAGCC.......
38


ON_LamHf2b2Ext
CCTCGACAGCGAAGTGCACAGAGCGCTTTG...............
30


ON_LamHf2dBrg(rc)
CCTCGACAGCTAAGTGCACAGAGCGCTTTGACTCAGCC.......
38


ON_LamHf2dExt
CCTCGACAGCGAAGTGCACAGAGCGCTTTG...............
30


ON_LamHf31Brg(rc)
CCTCGACAGCGAAGTGCACAGAGCGAATTGACTCAGCC.......
38


ON_LamHf31Ext
CCTCGACAGCGAAGTGCACAGAGCGAATTG...............
30


ON_LamHf3rBrg(rc)
CCTCGACAGCGAAGTGCACAGTACGAATTGACTCAGCC.......
38


ON_LamHf3rExt
CCTCGACAGCGAAGTGCACAGTACGAATTG...............
30


ON_lamPlePCR
CCTCGACAGCGAAGTGCACAG........................
21


Consensus
















TABLE 32





Oligonucleotides used to make SSDNA locally


double-stranded


(SEQ ID NOS 548-552,


respectively in order of appearance)















Adapters (8)








H43HF3.1?02#1 
5'-cc gtg tat tac tgt gcg aga g-3'


H43.77.97.1-03#2
5'-ct gtg tat tac tgt gcg aga g-3'


H43.77.97.323#22
5'-cc gta tat tac tgt gcg aaa g-3'


H43.77.97.330#23
5'-ct gtg tat tac tgt gcg aaa g-3'


H43.77.97.439#44
5'-ct gtg tat tac tgt gcg aga c-3'


H43.77.97.551#48
5'-cc atg tat tac tgt gcg aga c-3'
















TABLE 33





Bridge/extender pairs















Bridges (2)








H43.XABrl
(SEQ ID NO: 553)







5′ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagat


gaacagCTTtAGggctgaggacaCTGCAGtctactattgtgcgaga-3′ 


 








H43.XABr2
(SEQ ID NO: 554)







5′ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagat


gaacagCTTtAGggctgaggacaCTGCAGtctactattgtgcgaaa-3′


 


Extender








H43.XAExt
(SEQ ID NO: 555)







5′ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTcATcTgcAAgTA


gAgAgTATTcTTAgAgTTgTcTcTAgATcAcTAcAcc-3′
















TABLE 34





PCR primers















Primers








H43.XAPCR2
gactgggTgTAgTgATcTAg (SEQ ID NO:556)


 


Hucmnest
cttttctttgttgccgttggggtg (SEQ ID NO:557)
















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 20×



72 degrees C.
1 minute




72 degrees C.
7 minutes




4 degrees C.
hold










Reagents (100 ul reaction):











Template
5ul ligation mix




10× PCR buffer
1×




Taq
5U




dNTPs
200 uM each




MgCl2
2 mM




H43.XAPCR2-biotin
400 nM




Hucmnest
200 nM

















TABLE 36







!  Annotated sequence of CJR DY3F7(CJR-A05) 10251 bases


! 


! Non-cutters


!









!BclI Tgatca
BsiWI Cgtacg
BssSI Cacgag


!BstZl7I GTAtac
BtrI CACgtg
EcoRV GATatc


!FseI GGCCGGcc
HpaI GTTaac
MluI Acgcgt


!PmeI GTTTaaac
PmlI CACgtg
PpuMI RGgwccy


!RsrII CGgwccg
SapI GCTCTTC
SexAI Accwggt


!SgfI GCGATcgc
SgrAI CRccggyg
SphI GCATGc


!StuI AGGcct
XmaI Cccggg








! 


! cutters


! 


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


! 












!End of genes II and X

829





!Start gene V

843





!BsrGI Tgtaca
1
1021





!BspMI Nnnnnnnnngcaggt
3
1104
5997
9183








 (SEQ ID NO: 558)












!-′′- ACCTGCNNNNn
1
2281










 (SEQ ID NO: 559)












!End of gene V

1106





!Start gene VII

1108





!BsaBI GATNNnnatc
2
1149
3967









 (SEQ ID NO: 560)












!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





!Start gene III

1578





!EagI Cggccg
2
1630
8905




!XbaI Tctaga
2
1643
8436




!KasI Ggcgcc
4
1650
8724
9039
9120


!BsmI GAATGCN
2
1769
9065




!BseRI GAGGAGNNNNNNNNNN
2
2031
8516









(SEQ ID NO: 561)












!-′′- NNnnnnnnnnctcctc
2
7603
8623









(SEQ ID NO: 562)












! AlwNI CAGNNNctg
3
2210
8072
8182



! BspDI ATcgat
2
2520
9883




! NdeI CAtatg
3
2716
3796
9847



! End gene III

2846





! Start gene VI

2848





! AfeI AGCgct
1
3032





! End gene VI

3187





! Start gene I

3189





! Earl CTCTTCNnnn
2
4067
9274









(SEQ ID NO: 563)












! -′′-Nnnnngaagag
2
6126
8953









(SEQ ID NO: 564)












! PacI TTAATtaa
1
4125





! Start gene IV

4213





! End gene I

4235





! BsmFI Nnnnnnnnnnnnnnngtccc
2
5068
9515









(SEQ ID NO: 565)












! MscI TGGcca
3
5073
7597
9160



! PsiI TTAtaa
2
5349
5837




! End gene IV

5493





! Start on

5494





! NgoMIV Gccggc
3
5606
8213
9315



! BanII GRGCYc
4
5636
8080
8606 
8889


! DraIII CACNNNgtg
1
5709





! DrdI GACNNNNnngtc
1
5752










(SEQ ID NO: 566)












! AvaI Cycgrg
2
5818
7240




! PvuII CAGctg
1
5953





! BsmBI CGTCTCNnnnn
3
5964
8585
9271








(SEQ ID NO: 567)












! End onregion

5993





! BamHI Ggatcc
1
5994





! HindIII Aagctt
3
6000
7147
7384



! BciVI GTATCCNNNNNN
1
6077










(SEQ ID NO: 568)












! Start bla

6138





! Eco57I CTGAAG
2
6238
7716




! SpeI Actagt
1
6257





! BcgI gcannnnnntcg
1
6398










(SEQ ID NO: 569)












! ScaI AGTact
1
6442





! PvuI CGATcg
1
6553





! FspI TGCgca
1
6700





! BglI GCCNNNNnggc
3
6801
8208
8976








(SEQ ID NO:  570)












! BsaI GGTCTCNnnnn
1
6853










(SEQ ID NO: 571)












! AhdI GACNNNnngtc
1
6920










(SEQ ID NO:  572)












! Eam1105I GACNNNnngtc
1
6920










(SEQ ID NO: 573)












! End bla

6998





! AccI GTmkac
2
7153
8048




! HincII GTYrac
1
7153





! SalI Gtcgac
1
7153





! XhoI Ctcgag
1
7240





! Start PlacZ region

7246





! End PlacZ region

7381





! Pf1MI CCANNNNntgg
1
7382










(SEQ ID NO: 574)












! RBS1

7405





! start M13-iii signal seq for LC

7418





! ApaLI Gtgcac 1

7470





! end M13-iii signal seq

7471





! Start light chain kappa L20:JK1

7472





! Pf1FI GACNnngtc
3
7489
8705 
9099



! SbfI CCTGCAgg
1
7542





! PstI CTGCAg
1
7543





! KpnI GGTACc
1
7581





! XcmI CCANNNNNnnnntgg
2
7585
9215









(SEQ ID NO: 575)












! NsiI ATGCAt
2
7626
9503




! BsgI ctgcac
1
7809





! BbsI gtcttc
2
7820 
8616




! BlpI GCtnagc
1
8017





! EspI GCtnagc
1
8017





! Eco0109I RGgnccy
2
8073 
8605




! Ecl136I GAGctc
1
8080





! SacI GAGCTc
1
8080





! End light chain

8122





! AscI GGcgcgcc
1 
8126





! BssHII Gcgcgc
1 
8127





! RBS2

8147





! SfiI GGCCNNNNnggcc
1 
8207










(SEQ ID NO: 576)












! NcoI Ccatgg
1
8218





! Start 3-23, FR1

8226





! MfeI Caattg
1
8232





! BspEI Tccgga
1 
8298





! Start CDR1

8316





! Statt FR2

8331





! BstXI CCANNNNNntgg
2 
8339 
8812









(SEQ ID NO: 577)












! EcoNI CCTNNnnnagg
2
8346 
8675









(SEQ ID NO: 578)












! Start FR3

8373





! XbaI Tctaga
2
8436
1643




! AflII Cttaag
1
8480





! Start CDR3

8520





! AatII GACGTc
1
8556





! Start FR4

8562





! PshAI GACNNnngtc
2
8573
9231









(SEQ ID NO:  579)












! BstEII Ggtnacc
1
8579





! Start CH1

8595





! ApaI GGGCCc
1
8606





! Bsp120I Gggccc
1
8606





! PspOMI Gggccc
1
8606





! AgeI Accggt
1
8699





! Bsu36I CCtnagg
2 
8770
9509




! End of CH1

8903





! NotI GCggccgc
1 
8904





! Start His6 tag

8913










(SEQ ID NO: 12)












! Start cMyc tag

8931





! Amber codon

8982





! NheI Gctagc
1
8985





! Start M13 III Domain
3
8997





! NruI TCGcga
1
9106





! BstBI TTcgaa
1
9197





! EcoRI Gaattc
1
9200





! XcmI CCANNNNNnnnntgg
1
9215










(SEQ ID NO:  580)












! BstAPI GCANNNNntgc
1
9337










(SEQ ID NO:  581)












! SacII CCGCgg
1
9365





! End IIIstump anchor

9455





! AvrII Cctagg
1
9462





! trp terminator 

9470





! SwaI ATTTaaat
1
9784





! Start gene II

9850





! BglII Agatct
1
9936










!----------------------------------------------------------------------


!(SEQ ID NO:  582)


     1 aat gct act act att agt aga att gat gcc acc ttt tca gct cgc gcc


!    gene ii continued


    49 cca aat gaa aat ata gct aaa cag gtt att gac cat ttg cga aat gta


    97 tct aat ggt caa act aaa tct act cgt tcg cag aat tgg gaa tca act


   145 gtt aTa tgg aat gaa act tcc aga cac cgt act tta gtt gca tat tta


   193 aaa cat gtt gag cta cag caT TaT att cag caa tta agc tct aag cca


   241 tcc gca aaa atg acc tct tat caa aag gag caa tta aag gta ctc tct


   289 aat cct gac ctg ttg gag ttt gct tcc ggt ctg gtt cgc ttt gaa gct


   337 cga att aaa acg cga tat ttg aag tct ttc ggg ctt cct ctt aat ctt


   385 ttt gat gca atc cgc ttt gct tct gac tat aat agt cag ggt aaa gac


   433 ctg att ttt gat tta tgg tca ttc tcg ttt tct gaa ctg ttt aaa gca


   481 ttt gag ggg gat tca ATG aat att tat gac gat tcc gca gta ttg gac


!                          Start gene x, ii continues


   529 gct atc cag tct aaa cat ttt act att acc ccc tct ggc aaa act tct


   577 ttt gca aaa gcc tct cgc tat ttt ggt ttt tat cgt cgt ctg gta aac


   625 gag ggt tat gat agt gtt gct ctt act atg cct cgt aat tcc ttt tgg


   673 cgt tat gta tct gca tta gtt gaa tgt ggt att cct aaa tct caa ctg


   721 atg aat ctt tct acc tgt aat aat gtt gtt ccg tta gtt cgt ttt att


   769 aac gta gat ttt tct tcc caa cgt cct gac tgg tat aat gag cca gtt


   817 ctt aaa atc gca TAA


!                      End X & II


   832 ggtaattca ca


!(SEQ ID NO: 626)   


!       M1             E5                  Q10                 T15


   843 ATG att aaa gtt gaa att aaa cca tct caa gcc caa ttt act act cgt


!      Start gene V


!


!      S17         S20                 P25                 E30


   891 tct ggt gtt tct cgt cag ggc aag cct tat tca ctg aat gag cag ctt


!


!              V35                 E40                 V45


   939 tgt tac gtt gat ttg ggt aat gaa tat ccg gtt ctt gtc aag att act


!


!          D50                 A55                 L60


   987 ctt gat gaa ggt cag cca gcc tat gcg cct ggt cTGATAC 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


!


!                      P85     K87 end of V


! 1083 ctg cgc ctc gtt ccg gct aag TAA C


!


  1108 ATG gag cag gtc gcg gat ttc gac aca att tat cag gcg atg


!      Start gene VII


! 


  1150 ata caa atc tcc gtt gta ctt tgt ttc gcg ctt ggt ata atc


! 


!                        VII and IX overlap.


!                        ..... S2 V3 L4 V5 (SEQ ID NO: 621)  S10


  1192 gct ggg ggt caa agA TGA gt gtt tta gtg tat tct ttT gcc tct ttc


gtt








!


!      .... stop of IX, IX and VIII overlap by four bases


  1301 ATG aaa aag tct tta gtc ctc aaa gcc tct gta gcc gtt gct acc ctc


!      Start signal sequence of viii.


!


  1349 gtt ccg atg ctg tct ttc gct gct gag ggt gac gat ccc gca aaa gcg


!                                  mature VIII --->


  1397 gcc ttt aac tcc ctg caa gcc tca gcg acc gaa tat atc ggt tat gcg


  1445 tgg gcg atg gtt gtt gtc att


  1466 gtc ggc gca act atc ggt atc aag ctg ttt aag


!


! bases 1499-1539 are probable promoter for iii


  1499 aaa ttc acc tcg aaa gca !  1515


!       ...........  −35  ..


!


  1517 agc tga taaaccgat acaattaaag gctccttttg


!                                         ..... −10   ...


!


  1552 gagccttttt ttt GGAGAt ttt !  S.D. uppercase, there may be 9 Ts


!


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


 (SEQ ID NO:  583)


!            M   K   K   L   L   F   A   I   P   L   V   V   P   F


  1574 caac GTG aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc ! 


1620


!


!       Y   S   G   A   A   E   S   H   L   D   G   A


  1620 tat tct ggc gCG GCC Gaa tca caT CTA GAc ggc gcc


!                   EagI....         XbaI....


!


! Domain 1 ------------------------------------------------------------


!           A   E   T   V   E   S   C   L   A


  1656     gct gaa act gtt gaa agt tgt tta gca


!       K   S   H   T   E   I   S   F   T   N   V   W   K   D   D   K


T


  1683 aaA Tcc cat aca gaa aat tca ttt aCT AAC GTC TGG AAA GAC GAC AAA


ACt


!


!       L   D   R   Y   A   N   Y   E   G   S   L   W   N   A   T   G


V


  1734 tta gat cgt tac gct aac tat gag ggC tgt ctg tgG AAT GCt aca ggc


gtt


!                                                    BsmI....


!


!       V   V   C   T   G   D   E   T   Q   C   Y   G   T   W   V   P


I


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


att


!


!       G   L   A   I   P   E   N


  1836 ggg ctt gct atc cct gaa aat


! 


!  Ll linker------------------------------------


!       E   G   G   G   S   E   G   G   G   S


  1857 gag ggt ggt ggc tct gag ggt ggc ggt tct


!


!       E   G   G   G   S   E   G   G   G   T


  1887 gag ggt ggc ggt tct gag ggt ggc ggt act


! 


!  Domain 2-------------------------------------


  1917 aaa cct cct gag tac ggt gat aca cct att ccg ggc tat act tat atc


aac


  1968 cct ctc gac ggc act tat ccg cct ggt act gag caa aac ccc gct aat


cct


  2019 aat cct tct ctt GAG GAG tct cag cct ctt aat act ttc atg ttt cag


aat


!                      BseRI..


  2070 aat agg ttc cga aat agg cag ggg gca tta act gtt tat acg ggc act


  2118 gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act cct


  2166 gta tca tca aaa gcc atg tat gac gct tac tgg aac ggt aaa ttC AGA


!                                                                AlwNI


  2214 GAC TGc gct ttc cat tct ggc ttt aat gaG gat TTa ttT gtt tgt gaa


!      AlwNI


  2262 tat caa ggc caa tcg tct gac ctg cct caa cct cct gtc aat gct


!


  2307 ggc ggc ggc tct


!  start L2


-------------------------------------------------------------


  2319 ggt ggt ggt tct


  2331 ggt ggc ggc tct


  2343 gag ggt ggt ggc tct gag gga ggc ggt tcc


  2373 ggt ggt ggc tct ggt    ! end L2


!


! Many published sequences of M13-derived phage have a longer linker


! than shown here by repeats of the EGGGS (SEQ ID NO:  589) motif two


more times.


! 


! Domain 3


(SEQ ID NO:  584)


-------------------------------------------------------------


!       S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A


  2388 tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct


!


!       M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G


  2436 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc


!


!       K   L   D   S   V   A   T   D   Y   G   A   A   M   D   G   F


  2484 aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc


!


!       I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D


  2532 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat


!


!       F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N


  2580 ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat


!


!       S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q


  2628 tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa


!


!       S   V   E   C   R   P   F   V   F   G   A   G   K   P   Y   E


  2676 tcg gtt gaa tgt cgc cct ttt gtc ttt Ggc gct ggt aaa cca tat gaa


!


!       F   S   I   D   C   D   K   I   N   L   F   R


  2724 ttt tct att gat tgt gac aaa ata aac tta ttc cgt


!                                                  End Domain 3


!


!       G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V  F140


  2760 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt


!      start transmembrane segment


!


!       S   T   F   A   N   I   L


  2808 tct acg ttt gct aac ata ctg


!


!       R   N   K   E   S


  2829 cgt aat aag gag tct TAA !  stop of iii


!     Intracellular anchor.


!         (SEQ ID NO:  585)


!          M1  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 ctc ggt


!         Start VI


!


  2894 ttc ctt ctg gta act ttg ttc ggc tat ctg ctt act ttt ctt aaa aag


  2942 ggc ttc ggt aag ata gct att gct att tca ttg ttt ctt gct ctt att


  2990 att ggg ctt aac tca att ctt gtg ggt tat ctc tct gat att agc gct


  3038 caa tta ccc tct gac ttt gtt cag ggt gtt cag tta att ctc ccg tct


  3086 aat gcg ctt ccc tgt ttt tat gtt att ctc tct gta aag gct gct att


  3134 ttc att ttt gac gtt aaa caa aaa atc gtt tct tat ttg gat tgg gat


!


!                M1   A2  V3      F5                 L10         G13


  3182 aaa TAA t ATG gct gtt tat ttt gta act ggc aaa tta ggc tct gga


!      end VI    Start gene I


!


(SEQ ID NO:  586)


!       K   T   L   V   S   V   G   K   I   Q   D   K   I   V   A


  3228 aag acg ctc gtt agc gtt ggt aag att cag gat aaa att gta gct


!


!       G   C   K   I   A   T   N   L   D   L   R   L   Q   N   L


  3273 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac ctc


!


!       P   Q   V   G   R   F   A   K   T   P   R   V   L   R   I


  3318 ccg caa gtc ggg agg ttc gct aaa acg cct cgc gtt ctt aga ata


!


!       P   D   K   P   S   I   S   D   L   L   A   I   G   R   G


  3363 ccg gat aag cct tct ata tct gat ttg ctt gct att ggg cgc ggt


!


!       N   D   S   Y   D   E   N   K   N   G   L   L   V   L   D


  3408 aat gat tcc tac gat gaa aat aaa aac ggc ttg ctt gtt ctc gat


!


!       E   C   G   T   W   F   N   T   R   S   W   N   D   K   E


  3453 gag tgc ggt act tgg ttt aat acc cgt tct tgg aat gat aag gaa


!


!       R   Q   P   I   I   D   W   F   L   H   A   R   K   L   G


  3498 aga cag ccg att att gat tgg ttt cta cat gct cgt aaa tta gga


!


!       W   D   I   I   F   L   V   Q   D   L   S   I   V   D   K


  3543 tgg gat att att ttt ctt gtt cag gac tta tct att gtt gat aaa


!


!       Q   A   R   S   A   L   A   E   H   V   V   Y   C   R   R


  3588 cag gcg cgt tct gca tta gct gaa cat gtt gtt tat tgt cgt cgt


!


!       L   D   R   I   T   L   P   F   V   G   T   L   Y   S   L


  3633 ctg gac aga att act tta cct ttt gtc ggt act tta tat tct ctt


!


!       I   T   G   S   K   M   P   L   P   K   L   H   V   G   V


  3678 att act ggc tcg aaa atg cct ctg cct aaa tta cat gtt ggc gtt


!


!       V   K   Y   G   D   S   Q   L   S   P   T   V   E   R   W


  3723 gtt aaa tat ggc gat tct caa tta agc cct act gtt gag cgt tgg


!


!       L   Y   T   G   K   N   L   Y   N   A   Y   D   T   K   Q


  3768 ctt tat act ggt aag aat ttg tat aac gca tat gat act aaa cag


!


!       A   F   S   S   N   Y   D   S   G   V   Y   S   Y   L   T


  3813 gct ttt tct agt aat tat gat tcc ggt gtt tat tct tat tta acg


!


!       P   Y   L   S   H   G   R   Y   F   K   P   L   N   L   G


  3858 cct tat tta tca cac ggt cgg tat ttc aaa cca tta aat tta ggt


!


!       Q   K   M   K   L   T   K   I   Y   L   K   K   F   S   R


  3903 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tct cgc


!


!       V   L   C   L   A   I   G   F   A   S   A   F   T   Y   S


  3948 gtt ctt tgt ctt gcg att gga ttt gca tca gca ttt aca tat agt


!


!       Y   I   T   Q   P   K   P   E   V   K   K   V   V   S   Q


  3993 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tct cag


!


!       T   Y   D   F   D   K   F   T   I   D   S   S   Q   R   L


  4038 acc tat gat ttt gat aaa ttc act att gac tct tct cag cgt ctt


!


!       N   L   S   Y   R   Y   V   F   K   D   S   K   G   K   L


  4083 aat cta agc tat cgc tat gtt ttc aag gat tct aag gga aaa TTA 


!                                                              PacI


!


!       I   N   S   D   D   L   Q   K   Q   G   Y   S   L   T   Y


  4128 ATT AAt agc gac gat tta cag aag caa ggt tat tca ctc aca tat


!     PacI


!


!      i  I   D   L   C   T   V   S   I   K   K   G   N   S   N   E


!     iv                                                       M1  K


  4173   att gat tta tgt act gtt tcc att aaa aaa ggt aat tca aAT Gaa 


!                                                             Start IV


! (SEQ ID NO:  527)


!     i    I   V   K   C   N   .End of I


!     iv    L3  L   N5  V   I7  N    F  V10


  4218    att gtt aaa tgt aat TAA T TTT GTT


!  IV continued.....


  4243 ttc ttg atg ttt gtt tca tca tct tct ttt gct cag gta att gaa atg


  4291 aat aat tcg cct ctg cgc gat ttt gta act tgg tat tca aag caa tca


  4339 ggc gaa tcc gtt att gtt tct ccc gat gta aaa ggt act gtt act gta


  4387 tat tca tct gac gtt aaa cct gaa aat cta cgc aat ttc ttt att tct


  4435 gtt tta cgt gcA aat aat ttt gat atg gtA ggt tcT aAC cct tcc atT


  4483 att cag aag tat aat cca aac aat cag gat tat att gat gaa ttg cca


  4531 tca tct gat aat cag gaa tat gat gat aat tcc gct cct tct ggt ggt


  4579 ttc ttt gtt ccg caa aat gat aat gtt act caa act ttt aaa att aat


  4627 aac gtt cgg gca aag gat tta ata cga gtt gtc gaa ttg ttt gta aag


  4675 tct aat act tct aaa tcc tca aat gta tta tct att gac ggc tct aat


  4723 cta tta gtt gtt agt gcT cct aaa gat att tta gat aac ctt cct caa


  4771 ttc ctt tcA act gtt gat ttg cca act gac cag ata ttg att gag ggt


  4819 ttg ata ttt gag gtt cag caa ggt gat gct tta gat ttt tca ttt gct


  4867 gct ggc tct cag cgt ggc act gtt gca ggc ggt gtt aat act gac cgc


  4915 ctc acc tct gtt tta tct tct gct ggt ggt tcg ttc ggt att ttt aat


  4963 ggc gat gtt tta ggg cta tca gtt cgc gca tta aag act aat agc cat


  5011 tca aaa ata ttg tct gtg cca cgt att ctt acg ctt tca ggt cag aag


  5059 ggt tct atc 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 agc gtt ttt cct gtt gca atg gct ggc


  5203 ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct


  5251 tct act cag gca agt gat gtt att act aat caa aga agt att gct aca


  5299 acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggc ctc act


  5347 gat tat aaa aac act tct caG gat tct ggc gta ccg ttc ctg tct aaa


  5395 atc cct tta atc ggc ctc ctg ttt agc tcc cgc tct gat tcT aac gag


  5443 gaa agc acg tta tac gtg ctc gtc aaa gca acc ata gta cgc 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 gctttacggc


acctcgaccc


  5683 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat


agacggtttt


!                                  DraIII....


  5743 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc


aaactggaac


!               DrdI..........


  5803 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc


cgatttcgga


  5863 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg


cttgctgcaa


  5923 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact


ggtgaaaaga


!                                       PvuII.      BsmBI.


  5983 aaaaccaccc tGGATCC AAGCTT


!                  BamHI  HindIll (1/2)


!                  Insert carrying bla gene


  6006    gcaggtg gcacttttcg gggaaatgtg cgcggaaccc


  6043 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga


caataaccct


!                                           BciVI


6103 gataaatgct tcaataatat tgaaaaAGGA AGAgt


!                                RBS.?...


!    Start bla gene


6138 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca


ttt


  6189 tgc ctt cct gtt ttt gct cac cca gaa acg ctg gtg aaa gta aaa gat


gct


  6240 gaa gat cag ttg ggC gcA CTA GTg ggt tac atc gaa ctg gat ctc aac


agc


!                            SpeI....


!                       ApaLI & BssSI Removed


  6291 ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg


agc


  6342 act ttt aaa gtt ctg cta tgt GGC GcG Gta tta tcc cgt att gac gcc


ggg6393 caa gaG CAA CTC GGT CGc cgC ATA cAC tat tct cag aat gac ttg gtt


gAG


!             BcgI............


ScaI


  6444 TAC Tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga


gaa


!      ScaI.


  6495 tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac tta


ctt


  6546 ctg aca aCG ATC Gga gga ccg aag gag cta acc gct ttt ttg cac aac


atg


!               PvuI....


  6597 ggg gat cat gta act cgc ctt 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 cta tta act ggc gaa cta ctt act cta gct tcc cgg caa


caa


!       FspI....


! 


  6750 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc


tcg


  6801 GCC ctt ccG GCt ggc tgg ttt att gct gat aaa tct gga gcc ggt gag


cgt


!      BglI..........


  6852 gGG TCT Cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc


cgt


!       BsaI....


  6903 atc gta gtt atc tac acG ACg ggg aGT Cag gca act atg gat gaa cga


aat


!                            AhdI...........


  6954 aga cag atc gct gag ata  ggt gcc tca ctg att aag cat tgg TAA ctgt 


!                                                               stop


  7003 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt


taatttaaaa


  7063 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa


cgtgagtttt


  7123 cgttccactg tacgtaagac cccc


  7147 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct


!      HindIII SalI..


!      (2/2)    HincII


  7183 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt


! 


! Start of Fab-display cassette, the Fab DSR-A05, selected for


! binding to a protein antigen.


! 


  7233 CCTGAcG CTCGAG


!      xBsu36I XhoI..


! 


! PlacZ promoter is in the following block


! 


  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: (Amino acid sequence is SEQ ID NO: 587)


!          1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!          M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y


  7418    gtg aaa aaa tta tta ttc gca att cct tta gtt gtt cct ttc tat


!


!         16  17  18          Start light chain (L20:JK1)


!          S   H   S   A   Q   D   I   Q   M   T   Q   S   P   A


  7463    tct cac aGT GCA Caa gac atc cag atg acc cag tct cca gcc


!                  ApaLI...


!                  Sequence supplied by extender............


!


!               T L S L


  7505         acc ctg tct ttg


!


!          S   P   G   E   R   A   T   L   S   C   R   A   S   Q   G


  7517    tct cca ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag Ggt


!


!          V   S   S   Y   L   A   W   Y   Q   Q   K   P   G   Q   A


  7562    gtt agc agc tac tta gcc tgg tac cag cag aaa cct ggc cag get


!


!          P   R   L   L   I   Y   D   A   S   S   R   A   T   G   I


  7607    ccc agg ctc ctc atc tat gAt gca tcc aAc agg gcc act ggc atc


!


!          P   A   R   F   S   G   S   G   P   G   T   D   F   T   L


  7652    cca gCc agg ttc agt ggc agt ggg Cct ggg aca gac ttc act ctc


!


!          T   I   S   S   L   E   P   E   D   F   A   V   Y   Y   C


  7697    acc atc agc agC ctA gag cct gaa gat ttt gca gtT tat tac tgt


!


!          Q   Q   R   S   W   H   P   W    T   F   G   Q   G   T   R


  7742    cag cag CGt aAc tgg cat ccg tgg  ACG TTC GGC CAA GGG ACC AAG


!


!          V   E   I   K   R   T   V   A   A   P   S   V   F   I   F


  7787    gtg gaa atc aaa cga act gtg gCT GCA Cca tct gtc ttc atc ttc


!                                      BsgI....


!


!          P   P   S   D   E   Q   L   K   S   G   T   A   S   V   V


  7832    ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg


!


!          C   L   L   N   N   F   Y   P   R   E   A   K   V   Q   W


  7877    tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg


!


!          K   V   D   N   A   L   Q   S   G   N   S   Q   E   S   V


  7922    aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc


!


!          T   E   R   D   S   K   D   S   T   Y   S   L   S   S   T


  7967    aca gag cgg gac agc aag gac agc acc tac agc ctc agc agc acc


!


!          L   T   L   S   K   A   D   Y   E   K   H   K   V   Y   A


  8012    ctg acG CTG AGC aaa gca gac tac gag aaa cac aaa gtc tac gcc


!               EspI.....


!


!          C   E   V   T   H   Q   G   L   S   S   P   V   T   K   S


  8057    tgc gaa gtc acc cat cag ggc ctG AGC TCg ccc gtc aca aag agc


!                                       SacI....


!


!          F   N   R   G   E   C   .   .


  8102    ttc aac agg gga gag tgt taa taa


!


  8126        GGCGCG CCaattctat ttcaaGGAGA cagtcata


!             AscI.....              RBS2.


!       (Amino acid sequence is SEQ ID NO:  588)


!          PelB signal sequence------(22 codons)----->


!           1   2   3   4   5   6   7   8   9  10  11  12  13  14  15


!          M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L


  8160    atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc


!


!         ...PelB signal------------> Start VH, FR1----------------->


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


!          A   A   Q   P   A   M   A   E   V   Q   L   L   E   S   G


  8205    gcG GCC cag ccG GCC atg gcc gaa gtt CAA TTG tta gag tct ggt


!           SfiI.............                 MfeI...


!                          NcoI....


!


!          31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!          G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A


  8250    ggc ggt ctt gtt cag cct ggt ggt tct tta cgt ctt tct tgc gct


!


!         ...FR1--------------------> CDR1--------------> FR2-------->


!          46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!          A   S   G   F   T   F   S   T   Y   E   M   R   W   V   R


  8295    gct TCC GGA ttc act ttc tct 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


!          Q   A   P   G   K   G   L   E   W   V   S   Y   I   A   P


  8340    CAa gct ccT GGt aaa ggt ttg gag tgg gtt tct tat atc gct cct


!  BstXI................


!


!       ...CDR2---------------------------------------------> FR3---->


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


!          S   G   G   D   T   A   Y   A   D   S   V   K   G   R   F


  8385    tct ggt ggc gat act gct tat gct gac tcc gtt aaa ggt cgc ttc


!


!          91  92  93  94  95  96  97  98  99 100 101 102 103 104 105


!          T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M


  8430    act atc TCT AGA gac aac tct aag aat act ctc tac ttg cag atg


!                 XbaI...


!                 Supplied by extender-------------------------------


!


!         -----------------------------------------FR3-------------->


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


!          N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   R


  8475    aac agC TTA AGg gct gag gac act gca gtc tac tat tgt gcg agg


!               AflII...


!         from extender--------------------------------->


!


!         CDR3-------------------------------------------------->


FR4-->


!         121 122 123 124 125 126 127 128 129 130 131 132 133 134 135


!          R   L   D   G   Y   I   S   Y   Y   Y   G   M   D   V   W


  8520    agg ctc gat ggc tat att tcc tac tac tac ggt atg GAC GTC tgg


!                                                         AatII..


!


!         136 137 138 139 140 141 142 143 144 145


!          G   Q   G   T   T   V   T   V   S   S


  8565    ggc caa ggg acc acG GTC ACC gtc tca agc


!                           BstEII...


!


!         CH1 of IgG1---------->


!          A   S   T   K   G   P   S   V   F   P   L   A   P   S   S


  8595    gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc


!


!          K   S   T   S   G   G   T   A   A   L   G   C   L   V   K


  8640    aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag


!


!          D   Y   F   P   E   P   V   T   V   S   W   N   S   G   A


  8685    gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc


!


!          L   T   S   G   V   H   T   F   P   A   V   L   Q   S   S


  8730    ctg acc agc ggc gtc cac acc ttc ccg gct gtc cta cag tCC TCA


!


Bsu36I....


!


!          G   L   Y   S   L   S   S   V   V   T   V   P   S   S   S


  8775    GGa ctc tac tcc ctc agc agc gta gtg acc gtg ccc tcc agc agc


!  Bsu36I....


!


!          L   G   T   Q   T   Y   I   C   N   V   N   H   K   P   S


  8820    ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc


!


!          N   T   K   V   D   K   K   V   E   P   K   S   C   A   A


  8865    aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt GCG GCC


!


NotI......


!


!          A   H   H   H   H   H   H   G   A   A   E   Q   K   L   I


  8910    GCa cat cat cat cac cat cac ggg gcc gca gaa caa aaa ctc atc


!  ..NotI...  H6 tag................. Myc-Tag........................


!


!          S   E   E   D   L   N   G   A   A   q   A   S   S   A


  8955    tca gaa gag gat ctg aat ggg gcc gca tag GCT AGC tct gct


!         Myc-Tag....................         ... NheI...


!                                            Amber


!


! III'stump


! Domain 3 of III


---------------------------------------------------------


!


!       S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A


  8997 agt ggc gac ttc gac tac gag aaa atg gct aat gcc aac aaa GGC GCC


!      tcc   t   t   t   t   t   a   g       a   c   t   t   g   g   t


!W.T.


!


KasI...(2/4)


!


!       M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G


  9045 atG ACT GAG AAC GCT GAC GAG aat gct ttg caa agc gat gcc aag ggt


!            c   a   t   c   t   a   c   g c a   g tct   c   t   a   c


!W.T.


!


!       K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F


  9093 aag tta gac agc gTC GCG Acc gac tat GGC GCC gcc ATC GAc ggc ttt


!        a c t   t tct       t   t   t   c   t   t   t       t   t   c


!W.T.


!                       NruI....           KasI...(3/4)


!


!       I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D


  9141 atc ggc gat gtc agt ggt tTG GCC Aac ggc aac gga gcc acc gga gac


!        t   t   c   t tcc   c c t   t   t   t   t   t   t   t   t   t


!W.T.


!                               MscI....(3/3)


!


!       F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N


  9189 ttc GCA GGT tcG AAT TCt cag atg gcC CAG GTT GGA GAT GGg gac aac


!        t   t   c   t       c   a       t   a   c   t   c   t   t   t


!W.T.


!          BspMI.. (2/2)                 XcmI................


!                    EcoRI...


!


!       S   P   L   M,  N   N   F   R   Q   Y   L   P   S   L   R   Q


  9237 agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg cag


!      tca   t t a       t   t   c c t   a   t t a   t   c   c   t   a


!W.T.


!


!       S   V   E   C   R   P   F   V   F   S   A   G   K   P   Y   E


  9285 agt gtc gag tgc cgt cca ttc gtt ttc tct gcc ggc aag cct tac gag


!      tcg   t   a   t   c   t   t   c   t agc   t   t   a   a   t   a


!W.T.


!


!       F   S   I   D   C   D   K   I   N   L   F   R


  9333 ttc aGC Atc gac TGC gat aag atc aat ctt ttC CGC


!        t tct   t   t   t   c   a   a   c t a   c   t  !W.T.


!           BstAPI........                       SacII...


!                                                 End Domain 3


!


!       G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V   F


  9369 GGc gtt ttc gct ttc ttg cta tac gtc gct act ttc atg tac gtt ttc


!        t   c   t   g   t c t t a   t   t   c   c   t       t   a   t


! W.T.


!      start transmembrane segment


!


!       S   T   F   A   N   I   L     R   N   K   E   S


  9417 aGC ACT TTC GCC AAT ATT TTA   Cgc aac aaa gaa agc


!      tct   g   t   t   c   a c g     t   t   g   g tct ! W.T.


!                                    Intracellular anchor.


!               .   .


  9453         tag tga tct CCT AGG


!                          AvrII..








!  End Fab cassette


  9503 ATGCAT CCTGAGG ccgat actgtcgtcg tcccctcaaa ctggcagatg


!      NsiI.. Bsu36I.(3/3)


  9551 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt


caatccgccg


  9611 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt


tgatgaaagc


  9671 tggctacagg aaggccagac gcgaattatt tttgatggcg ttcctattgg


ttaaaaaatg


  9731 agctgattta acaaaaattt aaTgcgaatt ttaacaaaat attaacgttt


acaATTTAAA


!


Swal...


  9791 Tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat


  9850 ATG att gac atg cta gtt tta cga tta ccg ttc atc gat tct ctt gtt


tgc


!      Start gene II


  9901 tcc aga ctc tca ggc aat gac ctg ata gcc ttt gtA GAT CTc tca aaa


ata


!                                                    BglII...


  9952 gct acc ctc tcc ggc atT aat tta tca gct aga acg gtt gaa tat cat


att


 10003 gat ggt gat ttg act gtc tcc ggc ctt tct cac cct ttt gaa tct tta


cct


 10054 aca cat tac tca ggc att gca ttt aaa ata tat gag ggt tct aaa aat


ttt


 10105 tat cct tgc gtt gaa ata aag gct tct ccc gca aaa gta tta cag ggt


cat


 10156 aat gtt ttt ggt aca acc gat tta gct tta tgc tct gag gct tta ttg


ctt


 10207 aat ttt gct aat tct ttg cct tgc ctg tat gat tta ttg gat gtt ! 


! gene II continues


!-------------------------End of Table--------------------------------
















TABLE 37





!  DNA seq of w.t. M13 gene iii


(Nucleotide sequenc is SEQ ID NO: 590;


Amino acid sequence is SEQ ID NO: 591)
















! 
1    2   3   4   5   6   7   8   9  10  11  12  13  14  15


! 
fM   K   K   L   L   F   A   I   P  L   V   V   P   F   Y


 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


!
S   H   S   A   E   T   V   E   S   C   L   A   K   P   H


 1624 
tct cac tcc gct gaa act gtt gaa agt tgt tta gca aaa ccc cat







! Signal sequence> Domain 1----------------------------------


 








!
31  32  33  34  35  36  37  38  39  40  41  42  43  44  45


!
T   E   N   S   F   T   N   V   W   K   D   D   K   T   L


 1669 
aca gaa aat tca ttt act aac gtc tgg aaa gac gac aaa act tta


!
Domain 1---------------------------------------------------


 


!
46  47  48  49  50  51  52  53  54  55  56  57  58  59  60


!
D   R   Y   A   N   Y   E   G   C   L   W   N   A   T   G


 1714
gat cgt tac gct aac tat gag ggt tgt ctg tgG AAT GCt aca ggc


!
                                          BsmI....


!
Domain 1---------------------------------------------------


 


!
61  62  63  64  65  66  67  68  69  70  71  72  73  74  75


!
V   V   V   C   T   G   D   E   T   Q   C   Y   G   T   W


 1759
gtt gta gtt tgt act ggt gac gaa act cag tgt tac ggt aca tgg


!
Domain 1---------------------------------------------------


 


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


!
V   P   I   G   L   A   I   P   E   N   E   G   G   G   S


 1804
gtt cct att ggg ctt gct atc cct gaa aat gag ggt ggt ggc tct


!
Domain 1------------------------------> Linker 1-----------


 


!
91  92  93  94  95  96  97  98  99  100 101 102 103 104 105


!
E   G   G   G   S   E   G   G   G    S   E   G   G   G   T


 1849 
gag ggt ggc ggt tct gag ggt ggc ggt tct gag ggt ggc ggt act


!
Linker 1-------------------------------------------------->


 


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


!
 K   P   P   E   Y   G   D   T   P   I   P   G   Y   T   Y


 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


!
 I   N   P   L   D   G   T   Y   P   P   G   T   E   Q   N


 1939 
atc aac cct ctc gac ggc act taT CCG CCt ggt act gag caa aac


!
                              EciI....


!
Domain 2---------------------------------------------------


 


!
136 137 138 139 140 141 142 143 144 145 146 147 148 149 150


!
 P   A   N   P   N   P   S   L   E   E   S   Q   P   L   N


 1984 
ccc gct aat cct aat cct tct ctt GAG GAG tct cag cct ctt aat


!
                                BseRI..


!
Domain 2---------------------------------------------------


 


!
151 152 153 154 155 156 157 158 159 160 161 162 163 164 165


!
 T   F   M   F   Q   N   N   R   F   R   N   R   Q   G   A


 2029 
act ttc atg ttt cag aat aat agg ttc cga aat agg cag ggg gca


!
Domain 2---------------------------------------------------


 


!
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180


!
 L   T   V   Y   T   G   T   V   T   Q   G   T   D   P   V


 2074 
tta act gtt tat acg ggc act gtt act caa ggc act gac ccc gtt


!
Domain 2---------------------------------------------------


 


!
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195


!
 K   T   Y   Y   Q   Y   T   P   V   S   S   K   A   M   Y


 2119 
aaa act tat tac cag tac act cct gta tca tca aaa gcc atg tat


!
Domain 2---------------------------------------------------


 


!
196 197 198 199 200 201 202 203 204 205 206 207 208 209 210


!
 D   A   Y   W   N   G   K   F   R   D   C   A   F   H   S


 2164 
gac gct tac tgg aac ggt aaa ttC AGa gaC TGc gct ttc cat tct


!
                              A1wNI.......


!
Domain 2---------------------------------------------------


 


!
211 212 213 214 215 216 217 218 219 220 221 222 223 224 225


!
 G   F   N   E   D   P   F   V   C   E   Y   Q   G   Q   S


 2209 
ggc ttt aat gaG GAT CCa ttc gtt tgt gaa tat caa ggc caa tcg


!
              BamHI...


!
Domain 2---------------------------------------------------


 


!
226 227 228 229 230 231 232 233 234 235 236 237 238 239 240


!
 S   D   L   P   Q   P   P   V   N   A   G   G   G   S   G


 2254 
tct gac ctg cct caa cct cct gtc aat gct ggc ggc ggc tct ggt


!
Domain 2------------------------------> Linker 2-----------


 


!
241 242 243 244 245 246 247 248 249 250 251 252 253 254 255


!
 G   G   S   G   G   G   S   E   G   G   G   S   E   G   G


 2299 
ggt ggt tct ggt ggc ggc tct gag ggt ggt ggc tct gag ggt ggc


!
Linker 2---------------------------------------------------


 


!
256 257 258 259 260 261 262 263 264 265 266 267 268 269 270


!
 G   S   E   G   G   G   S   E   G   G   G   S   G   G   G


 2344 
ggt tct gag ggt ggc ggc tct gag gga ggc ggt tcc ggt ggt ggc


!
Linker 2---------------------------------------------------


 


!
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285


 2389 
 S   G   S   G   D   F   D   Y   E   K   M   A   N   A   N



tct ggt tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat







!Linker 2>    Domain 3-------------------------------------------


 








!
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300


!
 K   G   A   M   T   E   N   A   D   E   N   A   L   Q   S


 2434 
aag ggg gct atg acc gaa aat gcc gat gaa aac gcg cta cag tct


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


 


!
301 302 303 304 305 306 307 308 309 310 311 312 313 314 315


!
 D   A   K   G   K   L   D   S   V   A   T   D   Y   G   A


 2479 
gac gct aaa ggc aaa ctt gat tct gtc gct act gat tac ggt gct


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


 


!
316 317 318 319 320 321 322 323 324 325 326 327 328 329 330


!
 A   I   D   G   F   I   G   D   V   S   G   L   A   N   G


 2524 
gct atc gat ggt ttc att ggt gac gtt tcc ggc ctt gct aat ggt


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


 


!
331 332 333 334 335 336 337 338 339 340 341 342 343 344 345


!
 N   G   A   T   G   D   F   A   G   S   N   S   Q   M   A


 2569 
aat ggt gct act ggt gat ttt gct ggc tct aat tcc caa atg gct


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


 


!
346 347 348 349 350 351 352 353 354 355 356 357 358 359 360


!
 Q   V   G   D   G   D   N   S   P   L   M   N   N   F   R


 2614 
caa gtc ggt gac ggt gat aat tca cct tta atg aat aat ttc cgt


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


 


!
361 362 363 364 365 366 367 368 369 370 371 372 373 374 375


!
 Q   Y   L   P   S   L   P   Q   S   V   E   C   R   P   F


 2659 
caa tat tta cct tcc ctc cct caa tcg gtt gaa tgt cgc cct ttt


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


 


!
376 377 378 379 380 381 382 383 384 385 386 387 388 389 390


!
 V   F   S   A   G   K   P   Y   E   F   S   I   D   C   D


 2704 
gtc ttt agc gct ggt aaa cca tat gaa ttt tct att gat tgt gac


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


 


!
391 392 393 394 395 396 397 398 399 400 401 402 403 404 405


!
 K   I   N   L   F   R   G   V   F   A   F   L   L   Y   V


 2749 
aaa ata aac tta ttc cgt ggt gtc ttt gcg ttt ctt tta tat gtt


!
Domain 3--------------> Transmembrane segment--------------


 


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


!
 A   T   F   M   Y   V   F   S   T   F   A   N   I   L   R


 2794 
gcc acc ttt atg tat gta ttt tct acg ttt gct aac ata ctg cgt


!
Transmembrane segment---------------------------------> ICA--


 


!
421 422 423 424 425


!
 N   K   E   S   .


 2839 
aat aag gag tct taa ! 2853


!
ICA----------->            


 







!------------------End of Table


----------------------------------------





ICA = intracellular anchor













TABLE 38





Whole mature III anchor M13-III derived anchor with recoded DNA















!








!
 1   2   3









!
 A   A   A
(SEQ ID NO: 594)










!
 1
GCG gcc gca
(SEQ ID NO: 593)








!
NotI . . .


!
 4   5   6   7   8   9  10  11  12  13  14  15  16  17


!
 H   H   H   H   H   H   G   A   A   E   Q   K   L   I









!
10
cat cat cat cac cat cac ggg gcc gca gaa caa aaa ctc atc







!








!        
18  19  20  21  22  23  24  25  26  27  28  29



 S   E   E   D   L   N   G   A   A   .   A   S









!
52
tca gaa gag gat ctg aat ggg gcc gca Tag GCT AGC







                                                NheI...


!








!
30  31  32  33  34  35  36  37  38  39



 D   I   N   D   D   R   M   A   S   T









 
88
GAT ATC aac gat gat cgt atg get tct act







! (ON_G37bot) [RC] 5′-c aac gat gat cgt atg gcG CAt Gct gcc gag aca g-3′


         EcoRV.. (SEQ ID NO:  592)


!        Enterokinase cleavage site. 






































































































































!
354 355 356 357 358 359 360 361 362 363


!
 Q   M   A   Q   V   G   D   G   D   N










1060
cag atg gcC CAG GTT GGA GAT GGg gac aac








!
  a       t   a   c   t   c   t   t   t ! W.T.


!
          XcmI. . . . . . . . . . . . . . . .


 


!
364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379


!
 S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q










1090
agt ccg ctt atg aac aac ttt aga cag tac ctt ccg tct ctt ccg cag








!
tca   t t a       t   t   c c t   a   t t a   t   c   c   t   a ! W.T.


 


!
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395


!
 S   V   E   C   R   P   F   V   F   S   A   G   K   P   Y   E










1138
agt gtc gag tgc cgt cca ttc gtt ttc tct gcc ggc aag cct tac gag








!
tcg   t   a   t   c   t   t   c   t agc   t   t   a   a   t   a ! W.T.


 


!
Domain 3------------------------------------- >


!
396 397 398 399 400 401 402 403 404 405 406 407


!
 F   S   I   D   C   D   K   I   N   L   F   R










1186
ttc aGC Atc gac TGC gat aag atc aat ctt ttC CGC








!
  t tct   t   t   t   c   a   a   c t a       t


!
    BstAPI                                SacII...


 


!
transmembrane segment------------->


!
408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423


!
 G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V   F










1222
GGc gtt ttc gct ttc ttg cta tac gtc gct act ttc atg tac gtt ttc








!
  t   c   t   g   t c t t a   t   t   c   c   t       t   a   t ! W.T.


 









!
424 425 426 427 428 429 430 
431 432 433 434 435


!
 S   T   F   A   N   I   L   
 R   N   K   E   S











1270
aGC ACT TTC GCC AAT ATT TTA 
Cgc aac aaa gaa agc









!
tct   g   t   t   c   a c g 
  t   t   g   g tct ! W.T.








!
                             Intracellular anchor.


 










1306
        tag tga tct CCT AGG








!
                    AvrII..













! End Fab cassette


!----------------------------End of Table----------------------------
















TABLE 39





 ONs to make deletions in III















! ONs for use with NheI








! 
                                           N


(SEQ ID NO: 595)



(ON_G29bot)
5'-c gTT gAT ATc gcT Agc cTA Tgc-3'     ! 22


! this is the reverse complement of
5'-gca tag gct agc gat atc aac g-3'













!  50


!  ONs for use with SphI G CAT Gc








(ON_X37bot)
5′-gAc TgT cTc ggc Agc ATg cgc CAT Acg ATc ATc gTT g-3′ ! 37


(SEQ ID NO: 599)



! 
        N D D R M A H A (SEQ ID NO:  601)


! (ON_X37bot) = [RC] 
5′-c aac gat gat cgt atg gcG CAt Gct gcc gag aca gtc-3′







(SEQ ID NO: 600)








!  50
















TABLE 40







Phage titers and enrichments of selections with


a DY3F31-based human Fab library













Output/input



Input (total cfu)
Output (total cfu)
ratio





R1-ox selected on
4,5 × 1012
3,4 × 105
7,5 × 10−8


phOx-BSA





R2-Strep selected
9,2 × 1012
3 × 108
3,3 × 10−5


on Strep-beads
















TABLE 41







Frequency of ELISA positives in


DY3F31-based Fab libraries












9E10/RAM-
Anti-CK/CL



Anti-M13 HRP
HRP
Gar-HRP





R2-ox (with IPTG induction)
18/44
10/44
10/44


R2-ox (without IPTG)
13/44
ND
ND


R3-strep (with IPTG)
39/44
38/44
36/44


R3-strep (without IPTG)
33/44
ND
ND

## Claims

1. A library comprising a collection of members of a family, the family comprising a diversity of antibodies or functional fragments thereof, wherein the antibodies or functional fragments thereof each comprise a VH CDR1, a VH CDR2, a VH CDR3 and framework regions and the antibodies or functional fragments are encoded by DNA sequences comprising sequences encoding
(a) the VH CDR1, wherein the VH CDR1 comprises the amino acid sequence according to the formula -X1-Y-X2-M-X3- (SEQ ID NO:636), wherein X1, X2, and X3 are independently selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y,
(b) the VH CDR2, wherein the VH CDR2 comprises the amino acid sequence according to the formula X4-I-X5-X6-S-G-G-X7-T-X8-Y-A-D-S-V-K-G- (SEQ ID NO:637), wherein X4 and X5 are independently selected from the group consisting of Y, R, W, V, G, and S, X6 is selected from the group consisting of P and S, and X7 and X8 are independently selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y,
(c) the VH CDR3, wherein the sequence encoding the VH CDR3 is from the CDR3 region of an immunoglobulin gene of a B cell, and
(d) the framework regions, wherein the framework regions comprises VH 3-23 framework regions or VH3-23 related germline framework regions.

2. The library according to claim 1, wherein the antibodies or functional fragments thereof further comprise an immunoglobulin light chain, and said DNA sequences further comprise a sequence encoding the immunoglobin light chain.

3. The library according to claim 2, wherein said sequence encoding an immunoglobin light chain is from a B cell.

4. The library according to claim 3, wherein said B cell is from a blood sample from an autoimmune patient.

5. The library according to claim 4, wherein the autoimmune patient is diagnosed with a disorder selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis.

6. The library according to claim 1, wherein said antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

7. The library according to claim 1, wherein the diversity of antibodies or functional fragments thereof is displayed on genetic packages.

8. The library according to claim 7, wherein said genetic packages are M13 phage.

9. The library according to claim 8, wherein said DNA sequences are in a phage vector.

10. The library according to claim 9, wherein said phage vector comprises a wild-type gene iii and a truncated gene iii for display of the antibody or functional fragment thereof.

11. The library according to claim 8, wherein said DNA sequences are in a phagemid vector.

12. The library according to claim 8, wherein said displayed antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

13. The library according to claim 2, wherein the diversity of antibodies or functional fragments thereof is displayed on genetic packages.

14. The library according to claim 13, wherein said genetic packages are M13 phage.

15. The library according to claim 14, wherein said DNA sequences are in a phage vector.

16. The library according to claim 14, wherein said DNA sequences are in a phagemid vector.

17. The library according to claim 14, wherein said displayed antibodies or functional fragments thereof are linked via a short linker to the final portion of M13 gene III.

18. The library of claim 1, wherein the functional fragments are Fab fragments or scFv fragments.