# Patent

## Title
Position dependent recognition of GNN nucleotide triplets by zinc fingers

## Abstract

The specificity of binding of a zinc finger to a triplet or quadruplet nucleotide target subsite depends upon the location of the zinc finger in a multifinger protein and, hence, upon the location of its target subsite within a larger target sequence. The present disclosure provides zinc finger amino acid sequences for recognition of triplet target subsites having the nucleotide G in the 5′-most position of the subsite, that have been optimized with respect to the location of the subsite within the target site. Accordingly, the disclosure provides finger position-specific amino acid sequences for the recognition of GNN target subsites. This allows the construction of multi-finger zinc finger proteins with improved affinity and specificity for their target sequences, as well as enhanced biological activity.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application is a continuation of U.S. patent application Ser. No. 09/989,994 filed Nov. 20, 2001 now abandoned which is a continuation-in-part of U.S. patent application Ser. No. 09/535,088 filed Mar. 23, 2000, now abandoned, which application claims the benefit of U.S. provisional applications 60/126,238, filed Mar. 24, 1999, 60/126,239 filed Mar. 24, 1999, 60/146,595 filed Jul. 30, 1999 and 60/146,615 filed Jul. 30, 1999. U.S. patent application Ser. No. 09/989,994 filed Nov. 20, 2001 is also a continuation-in-part of U.S. patent application Ser. No. 09/716,637, filed Nov. 20, 2000, now U.S. Pat. No. 6,794,136. The disclosures of all of the aforementioned applications are hereby incorporated by reference in their entireties for all purposes.


SEQUENCE LISTING
Reference is made to the 947 page sequence listing which accompanies this patent application on compact disc form of 1.68 MB, submitted herewith in duplicate, entitled “Sequence Listing” and created on Sep. 22, 2005, which is herein incorporated by reference.
BACKGROUND
Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. An exemplary motif characterizing one class of these protein (C2H2 class) is -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His (where X is any amino acid) (SEQ. ID. No:1). A single finger domain is about 30 amino acids in length, and several structural studies have demonstrated that it contains an alpha helix containing the two invariant histidine residues and two invariant cysteine residues in a beta turn co-ordinated through zinc. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA-recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.
The x-ray crystal structure of Zif268, a three-finger domain from a murine transcription factor, has been solved in complex with a cognate DNA sequence and shows that each finger can be superimposed on the next by a periodic rotation. The structure suggests that each finger interacts independently with DNA over 3 base-pair intervals, with side-chains at positions −1, 2, 3 and 6 on each recognition helix making contacts with their respective DNA triplet subsites. The amino terminus of Zif268 is situated at the 3′ end of the DNA strand with which it makes most contacts. Some zinc fingers can bind to a fourth base in a target segment. If the strand with which a zinc finger protein makes most contacts is designated the target strand, some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the nontarget strand. The fourth base is complementary to the base immediately 3′ of the three base subsite.
The structure of the Zif268-DNA complex also suggested that the DNA sequence specificity of a zinc finger protein might be altered by making amino acid substitutions at the four helix positions (−1, 2, 3 and 6) on each of the zinc finger recognition helices. Phage display experiments using zinc finger combinatorial libraries to test this observation were published in a series of papers in 1994 (Rebar et al., Science 263, 671-673 (1994); Jamieson et al., Biochemistry 33, 5689-5695 (1994); Choo et al, PNAS 91, 11163-11167 (1994)). Combinatorial libraries were constructed with randomized side-chains in either the first or middle finger of Zif268 and then used to select for an altered Zif268 binding site in which the appropriate DNA sub-site was replaced by an altered DNA triplet. Further, correlation between the nature of introduced mutations and the resulting alteration in binding specificity gave rise to a partial set of substitution rules for design of ZFPs with altered binding specificity.
Greisman & Pabo, Science 275, 657-661 (1997) discuss an elaboration of the phage display method in which each finger of a Zif268 was successively randomized and selected for binding to a new triplet sequence. This paper reported selection of ZFPs for a nuclear hormone response element, a p53 target site and a TATA box sequence.
A number of papers have reported attempts to produce ZFPs to modulate particular target sites. For example, Choo et al., Nature 372, 645 (1994), report an attempt to design a ZFP that would repress expression of a bcr-abl oncogene. The target segment to which the ZFPs would bind was a nine base sequence 5′GCA GAA GCC3′ chosen to overlap the junction created by a specific oncogenic translocation fusing the genes encoding bcr and abl. The intention was that a ZFP specific to this target site would bind to the oncogene without binding to abl or bcr component genes. The authors used phage display to screen a mini-library of variant ZFPs for binding to this target segment. A variant ZFP thus isolated was then reported to repress expression of a stably transfected bcr-able construct in a cell line.
Pomerantz et al., Science 267, 93-96 (1995) reported an attempt to design a novel DNA binding protein by fusing two fingers from Zif268 with a homeodomain from Oct-1. The hybrid protein was then fused with a transcriptional activator for expression as a chimeric protein. The chimeric protein was reported to bind a target site representing a hybrid of the subsites of its two components. The authors then constructed a reporter vector containing a luciferase gene operably linked to a promoter and a hybrid site for the chimeric DNA binding protein in proximity to the promoter. The authors reported that their chimeric DNA binding protein could activate expression of the luciferase gene.
Liu et al., PNAS 94, 5525-5530 (1997) report forming a composite zinc finger protein by using a peptide spacer to link two component zinc finger proteins each having three fingers. The composite protein was then further linked to transcriptional activation domain. It was reported that the resulting chimeric protein bound to a target site formed from the target segments bound by the two component zinc finger proteins. It was further reported that the chimeric zinc finger protein could activate transcription of a reporter gene when its target site was inserted into a reporter plasmid in proximity to a promoter operably linked to the reporter.
Choo et al., WO 98/53058, WO98/53059, and WO 98/53060 (1998) discuss selection of zinc finger proteins to bind to a target site within the HIV Tat gene. Choo et al. also discuss selection of a zinc finger protein to bind to a target site encompassing a site of a common mutation in the oncogene ras. The target site within ras was thus constrained by the position of the mutation.
Previously-disclosed methods for the design of sequence-specific zinc finger proteins have often been based on modularity of individual zinc fingers; i.e., the ability of a zinc finger to recognize the same target subsite regardless of the location of the finger in a multi-finger protein. Although, in many instances, a zinc finger retains the same sequence specificity regardless of its location within a multi-finger protein; in certain cases, the sequence specificity of a zinc finger depends on its position. For example, it is possible for a finger to recognize a particular triplet sequence when it is present as finger 1 of a three-finger protein, but to recognize a different triplet sequence when present as finger 2 of a three-finger protein.
Attempts to address situations in which a zinc finger behaves in a non-modular fashion (i.e., its sequence specificity depends upon its location in a multi-finger protein) have, to date, involved strategies employing randomization of key binding residues in multiple adjacent zinc fingers, followed by selection. See, for example, Isalan et al. (2001) Nature Biotechnol. 19:656-660. However, methods for rational design of polypeptides containing non-modular zinc fingers have not heretofore been described.
SUMMARY
The present disclosure provides compositions comprising and methods involving position dependent recognition of GNN nucleotide triplets by zinc fingers.
Thus, provided herein is a zinc finger protein that binds to a target site, said zinc finger protein comprising a first (F1), a second (F2), and a third (F3) zinc finger, ordered F1, F2, F3 from N-terminus to C-terminus, said target site comprising, in 3′ to 5′ direction, a first (S1), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S3 comprises GGG, F3 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:1506); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S1 comprises GCA, F1 comprises the amino acid sequence QSGSLTR (SEQ ID NO:342); if S2 comprises GCA, F2 comprises QSGDLTR (SEQ ID NO:220); if S3 comprises GCA, F3 comprises QSGDLTR (SEQ ID NO:220); if S1 comprises GCG, F1 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S2 comprises GCG, F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S1 comprises GCC, F1 comprises the amino acid sequence ERGTLAR (SEQ ID NO:131); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S3 comprises GTG, F3 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184).
Also provided are methods of designing a zinc finger protein comprising a first (F1), a second (F2), and a third (F3) zinc finger, ordered F1, F2, F3 from N-terminus to C-terminus that binds to a target site comprising, in 3′ to 5′ direction, a first (S1), a second (S2), and a third (S3) target subsite, each target subsite having the nucleotide sequence GNN, the method comprising the steps of (a) selecting the F1 zinc finger such that it binds to the S1 target subsite, wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLRT (SEQ ID NO:1506); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S1 comprises GCA, F1 comprises QSGSLTR (SEQ ID NO:342); if S1 comprises GCG, F1 comprises RSDDLTR (SEQ ID NO:188); if S2 comprises GCG, F2 comprises RSDDLQR (SEQ ID NO:1844); if S1 comprises GCC, F1 comprises ERGTLAR (SEQ ID NO:131); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); (b) selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); and (c) selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGG, F3 comprises RSDHLSR (SEQ ID NO:229); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S3 comprises GTG, F3 comprises RSDALTR (SEQ ID NO:153); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184);
thereby designing a zinc finger protein that binds to a target site.
In certain embodiments of the zinc finger proteins and methods described herein, S1 comprises GAA and F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158). In other embodiments, S2 comprises GAA and F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801). In other embodiments, S3 comprises GAA and F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801). In other embodiments, S1 comprises GAG and F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130). In other embodiments, S2 comprises GAG and F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130). In other embodiments, S3 comprises GAG and F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231). In other embodiments, S1 comprises GAC and F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S2 comprises GAC and F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S3 comprises GAC and F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395). In other embodiments, S1 comprises GAT and F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765). In other embodiments, S2 comprises GAT and F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442). In other embodiments, S3 comprises GAT and F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377). In other embodiments, S1 comprises GGA and F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413). In other embodiments, S2 comprises GGA and F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287). In other embodiments, S3 comprises GGA and F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287). In other embodiments, S1 comprises GGG and F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127). In other embodiments, S2 comprises GGG and F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229). In other embodiments, S3 comprises GGG and F3 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229). In other embodiments, S1 comprises GGC and F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:705). In other embodiments, S2 comprises GGC and F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092). In other embodiments, S1 comprises GGT and F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835). In other embodiments, S2 comprises GGT and F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201). In other embodiments, S3 comprises GGT and F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425). In other embodiments, S1 comprises GCA and F1 comprises the amino acid sequence QSGSLTR (SEQ ID NO:342). In other embodiments, S2 comprises GCA and F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220). In other embodiments, S3 comprises GCA and F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220). In other embodiments, S1 comprises GCG and F1 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188). In other embodiments, S2 comprises GCG and F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844). In other embodiments, S3 comprises GCG and F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188). In other embodiments, S1 comprises GCC and F1 comprises the amino acid sequence ERGTLAR (SEQ ID NO:131). In other embodiments, S2 comprises GCC and F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417). In other embodiments, S3 comprises GCC and F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417). In other embodiments, S1 comprises GCT and F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450). In other embodiments, S2 comprises GCT and F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450). In other embodiments, S3 comprises GCT and F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132). In other embodiments, S1 comprises GTA and F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398). In other embodiments, S2 comprises GTA and F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339). In other embodiments, S1 comprises GTG and F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153). In other embodiments, S2 comprises GTG and F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237). In other embodiments, S3 comprises GTG and F3 comprises the amino acid sequence RSDALTR (SEQ ID NO:153). In other embodiments, S1 comprises GTC and F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184). In other embodiments, S2 comprises GTC and F2 comprises the amino acid sequence DRSALAR (SEQ ID NO:184). In other embodiments, S3 comprises GTC and F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184).
Also provided are polypeptides comprising any of zinc finger proteins described herein. In certain embodiments, the polypeptide further comprises at least one functional domain. Also provided are polynucleotides encoding any of the polypeptides described herein. Thus, also provided are nucleic acid encoding zinc fingers, including all of the zinc fingers described above.
Also provided are segments of a zinc finger comprising a sequence of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding any of these segments and zinc fingers comprising the same.
Also provided are zinc finger proteins comprising first, second and third zinc fingers. The first, second and third zinc fingers comprise respectively first, second and third segments of seven contiguous amino acids as shown herein. Also provided are nucleic acids encoding such zinc finger proteins.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows results of site selection analysis of two representative zinc finger proteins (leftmost 4 columns) and measurements of binding affinity for each of these proteins to their intended target sequences and to variant target sequences. (rightmost 3 columns). Analysis of ZFP1 is shown in the upper portion of the figure and analysis of ZFP2 is shown in the lower portion of the figure. For the site selection analyses, the amino acid sequences of residues −1 through +6 of the recognition helix of each of the three component zinc fingers (F3 (ZFP1, SEQ ID NO:130; ZFP2, SEQ ID NO:420), F2 (ZFP1, SEQ ID NO:1051; ZFP2, SEQ ID NO:889) and F1 (ZFP1, SEQ ID NO:395; ZFP2, SEQ ID NO:685)) are shown across the top row; the intended target sequence (divided into finger-specific target subsites) is shown across the second row, and a summary of the sequences bound is shown in the third row. Data for F3 is shown in the second column, data for F2 is shown in the third column, and data for F1 is shown in the third column.
For the binding affinity analyses, the designed target sequence for each ZFP (“cognate”) and two related sequences (“Mt”) are shown (column 6), along with the Kd for binding of the ZFP to each of these sequences (column 7).
FIG. 2 shows amino acid sequences of zinc finger recognition regions (amino acids −1 through +6 of the recognition helix) that bind to each of the 16 GNN triplet subsites. Three amino acid sequences are shown for each trinucleotide subsite; these correspond to optimal amino acid sequences for recognition of the subsite from each of the three positions (finger 1, F1 (SEQ ID NOS:688, 2534, 676, 1769, 342, 1450, 131, 158, 1765, 395, 1407, 2644, 705, 1398, 1733 & 184); finger 2, F2 (SEQ ID NOS:688, 229, 1446, 3051, 220, 1450, 417, 801, 1442, 395, 1824, 1201, 972, 3339, 1151 & 184); or finger 3, F3 (SEQ ID NOS:943, 229, 676, 1769, 220, 1365, 417, 801, 3525, 395, 1824, 1425, 972, 3592, 952 & 184)) in a three-finger zinc finger protein. Amino acid sequences are from N-terminal to C-terminal; nucleotide sequences are from 5′ to 3′.
Also shown are site selection results for each of the 48 position-dependent GNN-recognizing zinc fingers. These show the number of times a particular nucleotide was present, at a given position, in a collection of oligonucleotide sequences bound by the finger. For example, out of 15 oligonucleotides bound by a zinc finger protein with the amino acid sequence QSGHLAR (SEQ ID NO:413) present at the finger 1 (F1) position, 15 contained a G in the 5′-most position of the subsite, 15 contained a G in the middle position of the subsite, while, at the 3′-most position of the subsite, 10 contained an A, 3 contained a G and 2 contained a T. Accordingly, this particular amino acid sequence is optimal for binding a GGA triplet from the F1 position.
FIGS. 3A, 3B and 3C show site selection data indicating positional dependence of GCA-, GAT- and GGT-binding zinc fingers. The first and fourth (where applicable) rows of each figure show portions of the amino acid sequence of a designed zinc finger protein (F1 column, SEQ ID NOS:220, 1765, 1442, 835 & 1425; F2 column, SEQ ID NOS:220, 1765, 1442, 889, 1425; F3 column, SEQ ID NOS:220, 159, 377, 889 & 1425). Amino acid residues −1 through +6 of each α-helix are listed from left to right. The second and fifth (where applicable) rows show the target sequence, divided into three triplet subsites, one for each finger of the protein shown in the first and fourth (where applicable) rows, respectively. The third and sixth (where applicable) rows show the distribution of nucleotides in the oligonucleotides obtained by site selection with the proteins shown in the first and fourth (where applicable) rows, respectively. FIG. 3A shows data for fingers designed to bind GCA; FIG. 3B shows data for fingers designed to bind GAT; FIG. 3C shows data for fingers designed to bind GGT.
FIGS. 4A and 4B show properties of the engineered ZFP EP2C. FIG. 4A shows site selection data. The first row provides the amino acid sequences (F3, SEQ ID NO:1100; F2, SEQ ID NO:237; F1, SEQ ID NO:1450) of residues −1 through +6 of the recognition helices for each of the three zinc fingers of the EP2C protein. The second row shows the target sequence (5′ to 3′); with the distribution of nucleotides in the oligonucleotides obtained by site selection indicated below the target sequence.
FIG. 4B shows in vitro and in vivo assays for the binding specificity of EP2C. The first three columns show in vitro measurements of binding affinity of EP2C to its intended target sequence and several related sequences. The first column gives the name of each sequence (2C0 is the intended target sequence, compare to FIG. 4A). The second column shows the nucleotide sequence of various target sequences, with differences from the intended target sequence (2C0) highlighted. The third column shows the Kd (in nM) for binding of EP2C to each of the target sequences. Kds were determined by gel shift assays, using 2-fold dilution series of EP2C. The right side of the figure (fourth column and bar graph) shows relative luciferase activities (normalized to β-galactosidase levels) in stable cell lines in which expression of EP2C is inducible. Cells were co-transfected with a vector containing a luciferase coding region under the transcriptional control of the target sequence shown in the same row of the figure, and a control vector encoding β-galactosidase. Luciferase and β-galactosidase levels were measured after induction of EP2C expression. Triplicate samples were assayed and the standard deviations are shown in the bar graph. pGL3 is a luciferase-encoding vector lacking EP2C target sequences. 3B is another negative control, in which luciferase expression is under transcriptional control of sequences (3B) unrelated to the EP2C target sequence.



DEFINITIONS
A zinc finger DNA binding protein is a protein or segment within a larger protein that binds DNA in a sequence-specific manner as a result of stabilization of protein structure through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
Zinc finger proteins can be engineered to recognize a selected target sequence in a nucleic acid. Any method known in the art or disclosed herein can be used to construct an engineered zinc finger protein or a nucleic acid encoding an engineered zinc finger protein. These include, but are not limited to, rational design, selection methods (e.g., phage display) random mutagenesis, combinatorial libraries, computer design, affinity selection, use of databases matching zinc finger amino acid sequences with target subsite nucleotide sequences, cloning from cDNA and/or genomic libraries, and synthetic constructions. An engineered zinc finger protein can comprise a new combination of naturally-occurring zinc finger sequences. Methods for engineering zinc finger proteins are disclosed in co-owned WO 00/41566 and WO 00/42219; as well as in WO 98/53057; WO 98/53058; WO 98/53059 and WO 98/53060; the disclosures of which are hereby incorporated by reference in their entireties. Methods for identifying preferred target sequences, and for engineering zinc finger proteins to bind to such preferred target sequences, are disclosed in co-owned WO 00/42219.
A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.
A selected zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display.
The term naturally-occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. Generally, the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.
A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
A specific binding affinity between, for example, a ZFP and a specific target site means a binding affinity of at least 1×106 M−1.
The terms “modulating expression” “inhibiting expression” and “activating expression” of a gene refer to the ability of a zinc finger protein to activate or inhibit transcription of a gene. Activation includes prevention of subsequent transcriptional inhibition (i.e., prevention of repression of gene expression) and inhibition includes prevention of subsequent transcriptional activation (i.e., prevention of gene activation). Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels, changes in protein activity, changes in product levels, changes in downstream gene expression, changes in reporter gene transcription (luciferase, CAT, beta-galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); changes in signal transduction, phosphorylation and dephosphorylation, receptor-ligand interactions, second messenger concentrations (e.g., cGMP, cAMP, IP3, and Ca2+), cell growth, neovascularization, in vitro, in vivo, and ex vivo. Such functional effects can be measured by any means known to those skilled in the art, e.g., measurement of RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, calorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; cytokine release, and the like.
A “regulatory domain” refers to a protein or a protein subsequence that has transcriptional modulation activity. Typically, a regulatory domain is covalently or non-covalently linked to a ZFP to modulate transcription. Alternatively, a ZFP can act alone, without a regulatory domain, or with multiple regulatory domains to modulate transcription.
A D-able subsite within a target site has the motif 5′NNGK3′ (SEQ ID NO:4084). A target site containing one or more such motifs is sometimes described as a D-able target site. A zinc finger appropriately designed to bind to a D-able subsite is sometimes referred to as a D-able finger. Likewise a zinc finger protein containing at least one finger designed or selected to bind to a target site including at least one D-able subsite is sometimes referred to as a D-able zinc finger protein.
DETAILED DESCRIPTION
I. General
Tables 1-5 list a collection of nonnaturally occurring zinc finger protein sequences and their corresponding target sites. The first column of each table is an internal reference number. The second column lists a 9 or 10 base target site bound by a three-finger zinc finger protein, with the target sites listed in 5′ to 3′ orientation. The third column provides SEQ ID NOs for the target site sequences listed in column 2. The fourth, sixth and eighth columns list amino acid residues from the first, second and third fingers, respectively, of a zinc finger protein which recognizes the target sequence listed in the second column. For each finger, seven amino acids, occupying positions −1 to +6 of the finger, are listed. The numbering convention for zinc fingers is defined below. Columns 5, 7 and 9 provide SEQ ID NOs for the amino acid sequences listed in columns 4, 6 and 8, respectively. The final column of each table lists the binding affinity (i.e., the Kd in nM) of the zinc finger protein for its target site. Binding affinities are measured as described below.
Each finger binds to a triplet of bases within a corresponding target sequence. The first finger binds to the first triplet starting from the 3′ end of a target site, the second finger binds to the second triplet, and the third finger binds the third (i.e., the 5′-most) triplet of the target sequence. For example, the RSDSLTS finger (SEQ ID NO: 646) of SBS#201 (Table 2) binds to 5′TTG3′, the ERSTLTR finger (SEQ ID NO: 851) binds to 5′GCC3′ and the QRADLRR finger (SEQ ID NO: 1056) binds to 5′GCA3′.
Table 6 lists a collection of consensus sequences for zinc fingers and the target sites bound by such sequences. Conventional one letter amino acid codes are used to designate amino acids occupying consensus positions. The symbol “X” designates a nonconsensus position that can in principle be occupied by any amino acid. In most zinc fingers of the C2H2 type, binding specificity is principally conferred by residues −1, +2, +3 and +6. Accordingly, consensus sequence determining binding specificity typically include at least these residues. Consensus sequences are useful for designing zinc fingers to bind to a given target sequence. Residues occupying other positions can be selected based on sequences in Tables 1-5, or other known zinc finger sequences. Alternatively, these positions can be randomized with a plurality of candidate amino acids and screened against one or more target sequences to refine binding specificity or improve binding specificity. In general, the same consensus sequence can be used for design of a zinc finger regardless of the relative position of that finger in a multi-finger zinc finger protein. For example, the sequence RXDNXXR (SEQ ID NO:4060) can be used to design a N-terminal, central or C-terminal finger of three finger protein. However, some consensus sequences are most suitable for designing a zinc finger to occupy a particular position in a multi-finger protein. For example, the consensus sequence RXDHXXQ (SEQ ID NO:4055) is most suitable for designing a C-terminal finger of a three-finger protein.
II. Characteristics of Zinc Finger Proteins
Zinc finger proteins are formed from zinc finger components. For example, zinc finger proteins can have one to thirty-seven fingers, commonly having 2, 3, 4, 5 or 6 fingers. A zinc finger protein recognizes and binds to a target site (sometimes referred to as a target segment) that represents a relatively small subsequence within a target gene. Each component finger of a zinc finger protein can bind to a subsite within the target site. The subsite includes a triplet of three contiguous bases all on the same strand (sometimes referred to as the target strand). The subsite may or may not also include a fourth base on the opposite strand that is the complement of the base immediately 3′ of the three contiguous bases on the target strand. In many zinc finger proteins, a zinc finger binds to its triplet subsite substantially independently of other fingers in the same zinc finger protein. Accordingly, the binding specificity of zinc finger protein containing multiple fingers is usually approximately the aggregate of the specificities of its component fingers. For example, if a zinc finger protein is formed from first, second and third fingers that individually bind to triplets XXX, YYY, and ZZZ, the binding specificity of the zinc finger protein is 3′XXX YYY ZZZ5′.
The relative order of fingers in a zinc finger protein from N-terminal to C-terminal determines the relative order of triplets in the 3′ to 5′ direction in the target. For example, if a zinc finger protein comprises from N-terminal to C-terminal first, second and third fingers that individualy bind, respectively, to triplets 5′ GAC3′, 5′GTA3′ and 5′′GGC3′ then the zinc finger protein binds to the target segment 3′CAGATGCGG5′. If the zinc finger protein comprises the fingers in another order, for example, second finger, first finger, third finger, then the zinc finger protein binds to a target segment comprising a different permutation of triplets, in this example, 3′ATGCAGCGG5′ (see Berg & Shi, Science 271, 1081-1086 (1996)). The assessment of binding properties of a zinc finger protein as the aggregate of its component fingers may, in some cases, be influenced by context-dependent interactions of multiple fingers binding in the same protein.
Two or more zinc finger proteins can be linked to have a target specificity that is the aggregate of that of the component zinc finger proteins (see e.g., Kim & Pabo, PNAS 95, 2812-2817 (1998)). For example, a first zinc finger protein having first, second and third component fingers that respectively bind to XXX, YYY and ZZZ can be linked to a second zinc finger protein having first, second and third component fingers with binding specificities, AAA, BBB and CCC. The binding specificity of the combined first and second proteins is thus 3′XXXYYYZZZ______AAABBBCCC5′, where the underline indicates a short intervening region (typically 0-5 bases of any type). In this situation, the target site can be viewed as comprising two target segments separated by an intervening segment.
Linkage can be accomplished using any of the following peptide linkers. T G E K P: (SEQ. ID. No:2) (Liu et al., 1997, supra.); (G4S)n (SEQ. ID. No:3) (Kim et al., PNAS 93, 1156-1160 (1996); GGRRGGGS; (SEQ. ID. No:4) LRQRDGERP; (SEQ. ID. No:5) LRQKDGGGSERP; (SEQ. ID. No:6) LRQKD(G3S)2 ERP (SEQ. ID. No:7) Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling both DNA-binding sites and the peptides themselves or by phage display methods. In a further variation, noncovalent linkage can be achieved by fusing two zinc finger proteins with domains promoting heterodimer formation of the two zinc finger proteins. For example, one zinc finger protein can be fused with fos and the other with jun (see Barbas et al., WO 95/119431).
Linkage of two zinc finger proteins is advantageous for conferring a unique binding specificity within a mammalian genome. A typical mammalian diploid genome consists of 3×109 bp. Assuming that the four nucleotides A, C, G, and T are randomly distributed, a given 9 bp sequence is present  ̃23,000 times. Thus a ZFP recognizing a 9 bp target with absolute specificity would have the potential to bind to  ̃23,000 sites within the genome. An 18 bp sequence is present once in 3.4×1010 bp, or about once in a random DNA sequence whose complexity is ten times that of a mammalian genome.
A component finger of zinc finger protein typically contains about 30 amino acids and has the following motif (N-C):








(SEQ. ID. No: 8)









Cys-(X)2-4-Cys-X.X.X.X.X.X.X.X.X.X.X.X-His-(X)3-5-



His


 


                        −1 1 2 3 4 5 6 7






The two invariant histidine residues and two invariant cysteine residues in a single beta turn are co-ordinated through zinc (see, e.g., Berg & Shi, Science 271, 1081-1085 (1996)). The above motif shows a numbering convention that is standard in the field for the region of a zinc finger conferring binding specificity. The amino acid on the left (N-terminal side) of the first invariant His residues is assigned the number +6, and other amino acids further to the left are assigned successively decreasing numbers. The alpha helix begins at residue 1 and extends to the residue following the second conserved histidine. The entire helix is therefore of variable length, between 11 and 13 residues.
The process of designing or selecting a nonnaturally occurring or variant ZFP typically starts with a natural ZFP as a source of framework residues. The process of design or selection serves to define nonconserved positions (i.e., positions −1 to +6) so as to confer a desired binding specificity. One suitable ZFP is the DNA binding domain of the mouse transcription factor Zif268. The DNA binding domain of this protein has the amino acid sequence: YACPVESCDRRFSRSDELTRHIRIHTGQKP (F1) (SEQ. ID No:9) FQCRICMRNFSRSDHLTTHIRTHTGEKP (F2) (SEQ. ID. No:10) FACDICGRKFARSDERKRHTKIHLRQK (F3) SEQ. ID. No:11) and binds to a target 5′ GCG TGG GCG 3′ (SEQ ID No:12).
Another suitable natural zinc finger protein as a source of framework residues is Sp-1. The Sp-1 sequence used for construction of zinc finger proteins corresponds to amino acids 531 to 624 in the Sp-1 transcription factor. This sequence is 94 amino acids in length. The amino acid sequence of Sp-1 is as follows: PGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERP FMCTWSYCGKRFTRSDELQRHKRTHTGEKK FACPECPKRFMRSDHLSKHIKTHQNKKG (SEQ. ID. No:13) Sp-1 binds to a target site 5′GGG GCG GGG3′ (SEQ ID No: 14).
An alternate form of Sp-1, an Sp-1 consensus sequence, has the following amino acid sequence: meklrngsgd PGKKKQHACPECGKSFSKSSHLRAHQRTHTGERP YKCPECGKSFSRSDELQRHQRTHTGEKP YKCPECGKSFSRSDHLSKHQRTHQNKKG (SEQ. ID. NO:15) (lower case letters are a leader sequence from Shi & Berg, Chemistry and Biology 1, 83-89. (1995). The optimal binding sequence for the Sp-1 consensus sequence is 5′GGGGCGGGG3′ (SEQ ID NO: 16). Other suitable ZFPs are described below.
There are a number of substitution rules that assist rational design of some zinc finger proteins (see Desjarlais & Berg, PNAS 90, 2256-2260 (1993); Choo & Klug, PNAS 91, 11163-11167 (1994); Desjarlais & Berg, PNAS 89, 7345-7349 (1992); Jamieson et al., supra; Choo et al., WO 98/53057, WO 98/53058; WO 98/53059; WO 98/53060). Many of these rules are supported by site-directed mutagenesis of the three-finger domain of the ubiquitous transcription factor, Sp-1 (Desjarlais and Berg, 1992; 1993). One of these rules is that a 5′ G in a DNA triplet can be bound by a zinc finger incorporating arginine at position 6 of the recognition helix. Another substitution rule is that a G in the middle of a subsite can be recognized by including a histidine residue at position 3 of a zinc finger. A further substitution rule is that asparagine can be incorporated to recognize A in the middle of triplet, aspartic acid, glutamic acid, serine or threonine can be incorporated to recognize C in the middle of triplet, and amino acids with small side chains such as alanine can be incorporated to recognize T in the middle of triplet. A further substitution rule is that the 3′ base of triplet subsite can be recognized by incorporating the following amino acids at position −1 of the recognition helix: arginine to recognize G, glutamine to recognize A, glutamic acid (or aspartic acid) to recognize C, and threonine to recognize T. Although these substitution rules are useful in designing zinc finger proteins they do not take into account all possible target sites. Furthermore, the assumption underlying the rules, namely that a particular amino acid in a zinc finger is responsible for binding to a particular base in a subsite is only approximate. Context-dependent interactions between proximate amino acids in a finger or binding of multiple amino acids to a single base or vice versa can cause variation of the binding specificities predicted by the existing substitution rules.
The technique of phage display provides a largely empirical means of generating zinc finger proteins with a desired target specificity (see e.g., Rebar, U.S. Pat. No. 5,789,538; Choo et al., WO 96/06166; Barbas et al., WO 95/19431 and WO 98/543111; Jamieson et al., supra). The method can be used in conjunction with, or as an alternative to rational design. The method involves the generation of diverse libraries of mutagenized zinc finger proteins, followed by the isolation of proteins with desired DNA-binding properties using affinity selection methods. To use this method, the experimenter typically proceeds as follows. First, a gene for a zinc finger protein is mutagenized to introduce diversity into regions important for binding specificity and/or affinity. In a typical application, this is accomplished via randomization of a single finger at positions −1, +2, +3, and +6, and sometimes accessory positions such as +1, +5, +8 and +10. Next, the mutagenized gene is cloned into a phage or phagemid vector as a fusion with gene III of a filamentous phage, which encodes the coat protein pIII. The zinc finger gene is inserted between segments of gene III encoding the membrane export signal peptide and the remainder of pIII, so that the zinc finger protein is expressed as an amino-terminal fusion with pIII or in the mature, processed protein. When using phagemid vectors, the mutagenized zinc finger gene may also be fused to a truncated version of gene III encoding, minimally, the C-terminal region required for assembly of pIII into the phage particle. The resultant vector library is transformed into E. coli and used to produce filamentous phage which express variant zinc finger proteins on their surface as fusions with the coat protein pIII. If a phagemid vector is used, then the this step requires superinfection with helper phage. The phage library is then incubated with target DNA site, and affinity selection methods are used to isolate phage which bind target with high affinity from bulk phage. Typically, the DNA target is immobilized on a solid support, which is then washed under conditions sufficient to remove all but the tightest binding phage. After washing, any phage remaining on the support are recovered via elution under conditions which disrupt zinc finger-DNA binding. Recovered phage are used to infect fresh E. coli, which is then amplified and used to produce a new batch of phage particles. Selection and amplification are then repeated as many times as is necessary to enrich the phage pool for tight binders such that these may be identified using sequencing and/or screening methods. Although the method is illustrated for pIII fusions, analogous principles can be used to screen ZFP variants as pVIII fusions.
In certain embodiments, the sequence bound by a particular zinc finger protein is determined by conducting binding reactions (see, e.g., conditions for determination of Kd, infra) between the protein and a pool of randomized double-stranded oligonucleotide sequences. The binding reaction is analyzed by an electrophoretic mobility shift assay (EMSA), in which protein-DNA complexes undergo retarded migration in a gel and can be separated from unbound nucleic acid. Oligonucleotides which have bound the finger are purified from the gel and amplified, for example, by a polymerase chain reaction. The selection (i.e. binding reaction and EMSA analysis) is then repeated as many times as desired, with the selected oligonucleotide sequences. In this way, the binding specificity of a zinc finger protein having a particular amino acid sequence is determined.
Zinc finger proteins are often expressed with a heterologous domain as fusion proteins. Common domains for addition to the ZFP include, e.g., transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g. kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. A preferred domain for fusing with a ZFP when the ZFP is to be used for repressing expression of a target gene is a KRAB repression domain from the human KOX-1 protein (Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994). Preferred domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11, 4961-4968 (1992)).
An important factor in the administration of polypeptide compounds, such as the ZFPs, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.
For example, “membrane translocation polypeptides” have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)).
Examples of peptide sequences which can be linked to a ZFP, for facilitating uptake of ZFP into cells, include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs.
Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called “binary toxins”): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used in attempts to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993); Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNAS U.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem. 267:17186-17193 1992)).
Such subsequences can be used to translocate ZFPs across a cell membrane. ZFPs can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
III. Position Dependence of Subsite Recognition by Zinc Fingers
A number of the polypeptides disclosed herein have been characterized using the methods disclosed in U.S. Pat. No. 6,794,136; which matured from a grandparent of the present application (the disclosure of which is hereby incorporated by reference in its entirety); in particular with respect to the effect of their position, within a multi-finger protein, on their sequence specificity. The results of these investigations provide a set of zinc finger sequences that are optimized for recognition of certain triplet target subsites whose 5′-most nucleotide is a G (i.e., GNN triplet subsites). Thus, particular zinc finger sequences which recognize each of the GNN triplet subsites, from each position of a three-finger zinc finger protein, are provided. See FIG. 2. It will be clear to those of skill in the art that the optimized, position-specific zinc finger sequences disclosed herein for recognition of GNN target subsites are not limited to use in three-finger proteins. For example, they are also useful in six-finger proteins, which can be made by linkage of two three-finger proteins.
A number of zinc finger amino acid sequences which are reported to bind to target subsites in which the 5′-most nucleotide residue is G (i.e., GNN subsites) have recently been disclosed. Segal et al. (1999) Proc. Natl. Acad. Sci. USA 96:2758-2763; Drier et al. (2000) J. Mol. Biol. 303:489-502; U.S. Pat. No. 6,140,081. These GNN-binding zinc fingers were obtained by selection of finger 2 sequences from phage display libraries of three-finger proteins, in which certain amino acid residues of finger 2 had been randomized. Due to the manner in which they were selected, it is not clear whether these sequences would have the same target subsite specificity if they were present in the F1 and/or F3 positions.
Use of the methods and compositions disclosed herein has now allowed identification of specific zinc finger sequences that bind each of the 16 GNN triplet subsites, and for the first time, provides zinc finger sequences that are optimized for recognition of these triplet subsites in a position-dependent fashion. Moreover, in vivo studies of these optimized designs reveal that the functionality of a ZFP is correlated with its binding affinity to its target sequence. See Example 6, infra.
As a result of the discovery, disclosed herein, that sequence recognition by zinc fingers is position-dependent, it is clear that existing design rules will not, in and of themselves, be applicable to every situation in which it is necessary to construct a sequence-specific ZFP. The results disclosed herein show that many zinc fingers that are constructed based on design rules exhibit the sequence specificity predicted by those design rules only at certain finger positions. The position-specific zinc fingers disclosed herein are likely to function more efficiently in vivo and in cultured cells, with fewer nonspecific effects. Highly specific ZFPs, made using position-specific zinc fingers, will be useful tools in studying gene function and will find broad applications in areas as diverse as human therapeutics and plant engineering.
IV. Production of Zinc Finger Proteins
ZFP polypeptides and nucleic acids encoding the same can be made using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In addition, nucleic acids less than about 100 bases can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, Calif.). Similarly, peptides can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc.
Oligonucleotides can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either denaturing polyacrylamide gel electrophoresis or by reverse phase HPLC. The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).
Two alternative methods are typically used to create the coding sequences required to express newly designed DNA-binding peptides. One protocol is a PCR-based assembly procedure that utilizes six overlapping oligonucleotides (FIG. 1). Three oligonucleotides (oligos 1, 3, and 5 in FIG. 1) correspond to “universal” sequences that encode portions of the DNA-binding domain between the recognition helices. These oligonucleotides typically remain constant for all zinc finger constructs. The other three “specific” oligonucleotides (oligos 2, 4, and 6 in FIG. 1) are designed to encode the recognition helices. These oligonucleotides contain substitutions primarily at positions −1, 2, 3 and 6 on the recognition helices making them specific for each of the different DNA-binding domains.
The PCR synthesis is carried out in two steps. First, a double stranded DNA template is created by combining the six oligonucleotides (three universal, three specific) in a four cycle PCR reaction with a low temperature annealing step, thereby annealing the oligonucleotides to form a DNA “scaffold.” The gaps in the scaffold are filled in by high-fidelity thermostable polymerase, the combination of Taq and Pfu polymerases also suffices. In the second phase of construction, the zinc finger template is amplified by external primers designed to incorporate restriction sites at either end for cloning into a shuttle vector or directly into an expression vector.
An alternative method of cloning the newly designed DNA-binding proteins relies on annealing complementary oligonucleotides encoding the specific regions of the desired ZFP. This particular application requires that the oligonucleotides be phosphorylated prior to the final ligation step. This is usually performed before setting up the annealing reactions. In brief, the “universal” oligonucleotides encoding the constant regions of the proteins (oligos 1, 2 and 3 of above) are annealed with their complementary oligonucleotides. Additionally, the “specific” oligonucleotides encoding the finger recognition helices are annealed with their respective complementary oligonucleotides. These complementary oligos are designed to fill in the region which was previously filled in by polymerase in the above-mentioned protocol. The complementary oligos to the common oligos 1 and finger 3 are engineered to leave overhanging sequences specific for the restriction sites used in cloning into the vector of choice in the following step. The second assembly protocol differs from the initial protocol in the following aspects: the “scaffold” encoding the newly designed ZFP is composed entirely of synthetic DNA thereby eliminating the polymerase fill-in step, additionally the fragment to be cloned into the vector does not require amplification. Lastly, the design of leaving sequence-specific overhangs eliminates the need for restriction enzyme digests of the inserting fragment. Alternatively, changes to ZFP recognition helices can be created using conventional site-directed mutagenesis methods.
Both assembly methods require that the resulting fragment encoding the newly designed ZFP be ligated into a vector. Ultimately, the ZFP-encoding sequence is cloned into an expression vector. Expression vectors that are commonly utilized include, but are not limited to, a modified pMAL-c2 bacterial expression vector (New England BioLabs or an eukaryotic expression vector, pcDNA (Promega). The final constructs are verified by sequence analysis.
Any suitable method of protein purification known to those of skill in the art can be used to purify ZFPs (see, Ausubel, supra, Sambrook, supra). In addition, any suitable host can be used for expression, e.g., bacterial cells, insect cells, yeast cells, mammalian cells, and the like.
Expression of a zinc finger protein fused to a maltose binding protein (MBP-ZFP) in bacterial strain JM109 allows for straightforward purification through an amylose column (NEB). High expression levels of the zinc finger chimeric protein can be obtained by induction with IPTG since the MBP-ZFP fusion in the pMal-c2 expression plasmid is under the control of the tac promoter (NEB). Bacteria containing the MBP-ZFP fusion plasmids are inoculated into 2×YT medium containing 10 μM ZnCl2, 0.02% glucose, plus 50 μg/ml ampicillin and shaken at 37° C. At mid-exponential growth IPTG is added to 0.3 mM and the cultures are allowed to shake. After 3 hours the bacteria are harvested by centrifugation, disrupted by sonication or by passage through a french pressure cell or through the use of lysozyme, and insoluble material is removed by centrifugation. The MBP-ZFP proteins are captured on an amylose-bound resin, washed extensively with buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 μM ZnCl2, then eluted with maltose in essentially the same buffer (purification is based on a standard protocol from NEB). Purified proteins are quantitated and stored for biochemical analysis.
The dissociation constants of the purified proteins, e.g., Kd, are typically characterized via electrophoretic mobility shift assays (EMSA) (Buratowski & Chodosh, in Current Protocols in Molecular Biology pp. 12.2.1-12.2.7 (Ausubel ed., 1996)). Affinity is measured by titrating purified protein against a fixed amount of labeled double-stranded oligonucleotide target. The target typically comprises the natural binding site sequence flanked by the 3 bp found in the natural sequence and additional, constant flanking sequences. The natural binding site is typically 9 bp for a three-finger protein and 2×9 bp+intervening bases for a six finger ZFP. The annealed oligonucleotide targets possess a 1 base 5′ overhang which allows for efficient labeling of the target with T4 phage polynucleotide kinase. For the assay the target is added at a concentration of 1 nM or lower (the actual concentration is kept at least 10-fold lower than the expected dissociation constant), purified ZFPs are added at various concentrations, and the reaction is allowed to equilibrate for at least 45 min. In addition the reaction mixture also contains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.1 mM ZnCl2, 5 mM DTT, 10% glycerol, 0.02% BSA. (NB: in earlier assays poly d(IC) was also added at 10-100 μg/μl.)
The equilibrated reactions are loaded onto a 10% polyacrylamide gel, which has been pre-run for 45 min in Tris/glycine buffer, then bound and unbound labeled target is resolved by electrophoresis at 150V. (alternatively, 10-20% gradient Tris-HCl gels, containing a 4% polyacrylamide stacker, can be used) The dried gels are visualized by autoradiography or phosphorimaging and the apparent Kd is determined by calculating the protein concentration that gives half-maximal binding.
The assays can also include determining active fractions in the protein preparations. Active fractions are determined by stoichiometric gel shifts where proteins are titrated against a high concentration of target DNA. Titrations are done at 100, 50, and 25% of target (usually at micromolar levels).
V. Applications of Engineered Zinc Finger Proteins
ZPFs that bind to a particular target gene, and the nucleic acids encoding them, can be used for a variety of applications. These applications include therapeutic methods in which a ZFP or a nucleic acid encoding it is administered to a subject and used to modulate the expression of a target gene within the subject. See, for example, co-owned WO 00/41566. The modulation can be in the form of repression, for example, when the target gene resides in a pathological infecting microrganisms, or in an endogenous gene of the patient, such as an oncogene or viral receptor, that is contributing to a disease state. Alternatively, the modulation can be in the form of activation when activation of expression or increased expression of an endogenous cellular gene can ameliorate a diseased state. For such applications, ZFPs, or more typically, nucleic acids encoding them are formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)). The ZFPs, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
The dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose is determined by the efficacy and Kd of the particular ZFP employed, the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also is determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient
In other applications, ZFPs are used in diagnostic methods for sequence specific detection of target nucleic acid in a sample. For example, ZFPs can be used to detect variant alleles associated with a disease or phenotype in patient samples. As an example, ZFPs can be used to detect the presence of particular mRNA species or cDNA in a complex mixtures of mRNAs or cDNAs. As a further example, ZFPs can be used to quantify copy number of a gene in a sample. For example, detection of loss of one copy of a p53 gene in a clinical sample is an indicator of susceptibility to cancer. In a further example, ZFPs are used to detect the presence of pathological microorganisms in clinical samples. This is achieved by using one or more ZFPs specific to genes within the microorganism to be detected. A suitable format for performing diagnostic assays employs ZFPs linked to a domain that allows immobilization of the ZFP on an ELISA plate. The immobilized ZFP is contacted with a sample suspected of containing a target nucleic acid under conditions in which binding can occur. Typically, nucleic acids in the sample are labeled (e.g., in the course of PCR amplification). Alternatively, unlabelled probes can be detected using a second labelled probe. After washing, bound-labelled nucleic acids are detected.
ZFPs also can be used for assays to determine the phenotype and function of gene expression. Current methodologies for determination of gene function rely primarily upon either overexpression or removing (knocking out completely) the gene of interest from its natural biological setting and observing the effects. The phenotypic effects observed indicate the role of the gene in the biological system.
One advantage of ZFP-mediated regulation of a gene relative to conventional knockout analysis is that expression of the ZFP can be placed under small molecule control. By controlling expression levels of the ZFPs, one can in turn control the expression levels of a gene regulated by the ZFP to determine what degree of repression or stimulation of expression is required to achieve a given phenotypic or biochemical effect. This approach has particular value for drug development. By putting the ZFP under small molecule control, problems of embryonic lethality and developmental compensation can be avoided by switching on the ZFP repressor at a later stage in mouse development and observing the effects in the adult animal. Transgenic mice having target genes regulated by a ZFP can be produced by integration of the nucleic acid encoding the ZFP at any site in trans to the target gene. Accordingly, homologous recombination is not required for integration of the nucleic acid. Further, because the ZFP is trans-dominant, only one chromosomal copy is needed and therefore functional knock-out animals can be produced without backcrossing.
All references cited above are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
Example 1
Initial Design of Zinc Finger Proteins and Determination of Binding Affinity
Initial ZFP designs were based on existing design rules, correspondence regimes and ZFP directories, including those disclosed herein (see Tables 1-5) and also in WO 98/53058; WO 98/530059; WO 98/53060 and co-owned U.S. patent application Ser. No. 09/444,241. See also WO 00/42219. Amino acid sequences were conceptually designed using amino acids 532-624 of the human transcription factor Sp1 as a backbone. Polynucleotides encoding designed ZFPs were assembled using a Polymerase Chain Reaction (PCR)-based procedure that utilizes six overlapping oligonucleotides. PCR products were directly cloned cloning into the Tac promoter vector, pMal-c2 (New England Biolabs, Beverly, Mass.) using the KpnI and BamHI restriction sites. The encoded maltose binding protein-ZFP fusion polypeptides were purified according to the manufacturer's procedures (New England Biolabs, Beverly, Mass.). Binding affinity was measured by gel mobility-shift analysis. All of these procedures are described in detail in co-owned WO 00/41566 and WO 00/42219, as well as in Zhang et al. (2000) J. Biol. Chem. 275:33,850-33,860 and Liu et al. (2001) J. Biol. Chem. 276:11,323-11,334; the disclosures of which are hereby incorporated by reference in their entireties.
Example 2
Optimization of Binding Specificity by Site Selection
Designed ZFPs were tested for binding specificity using site selection methods disclosed in parent application U.S. Ser. No. 09/716,637 filed Nov. 20, 2000, now U.S. Pat. No. 6,794,136. Briefly, designed proteins were incubated with a population of labeled, double-stranded oligonucleotides comprising a library of all possible 9- or 10-nucleotide target sequences. Five nanomoles of labeled oligonucleotides were incubated with protein, at a protein concentration 4-fold above its Kd for its target sequence. The mixture was subjected to gel electrophoresis, and bound oligonucleotides were identified by mobility shift, and extracted from the gel. The purified bound oligonucleotides were amplified, and the amplification products were used for a subsequent round of selection. At each round of selection, the protein concentration was decreased by 2 fold. After 3-5 rounds of selection, amplification products were cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.), and the nucleotide sequences of approximately 20 clones were determined. The identities of the target sites bound by a designed protein were determined from the sequences and expressed as a compilation of subsite binding sequences.
Example 3
Comparison of Site Selection Results with Binding Affinity
To test the correlation between site selection results and the affinity of binding of a ZFP to various related targets, site selection experiments were conducted on 2 three-finger ZFPs, denoted ZFP1 and ZFP2, and the site selection results were compared with Kd measurements obtained from quantitative gel-mobility shift assays using the same ZFPs and target sites. Each ZFP was constructed, based on design rules, to bind to a particular nine-nucleotide target sequence (comprising 3 three-nucleotide subsites), as shown in FIG. 1. Site selection results and affinity measurements are also shown in FIG. 1. The site selection results showed that fingers 1 and 3 of both the ZFP1 and ZFP2 proteins preferentially selected their intended target sequences. However, the second finger of each ZFP preferentially selected subsites other than those to which they were designed to bind (e.g., F2 of ZFP1 was designed to bind TCG, but preferentially selected GTG; F2 of ZFP2 was designed to bind GGT, but preferentially selected GGA).
To confirm the site selection results, binding affinities of ZFP1 and ZFP2 were measured (see Example 1, supra), both to their original target sequences and to new target sequences reflecting the site selection results. For example, the Mt-1 sequence contains two base changes (compared to the original target sequence for ZFP1) which result in a change in the sequence of the finger 2 subsite to GTG, reflecting the preferred finger 2 subsite sequence obtained by site selection. In agreement with the site selection results, binding of ZFP1 to the Mt-1 sequence is approximately 4-fold stronger than its binding to the original target sequence (Kd of 12.5 nM compared to a Kd of 50 nM, see FIG. 1).
For ZFP2, the specificity of finger 2 for the 3′ base of its target subsite was tested, since, although this finger was designed to bind GGT, site selection indicated that it bound preferentially to GGA. Moreover, the site selection results predicted that finger 2 of ZFP2 would bind with approximately equal affinity to GGT and GGC. Accordingly, target sequences containing GGA (Mt-3) and GGC (Mt-4) at the finger 2 subsite were constructed, and binding affinities of ZFP2 to these target sequences, and to its original target sequence (containing GGT at the finger 2 subsite), were compared. In complete agreement with the site selection results, ZFP2 exhibited the strongest binding affinity for the target sequence containing GGA at the finger 2 subsite (Kd of 0.5 nM, FIG. 1), and its affinity for target sequences containing either GGT or GGC at the finger 2 subsite was approximately equal (Kd of 1 nM for both targets, FIG. 1). Accordingly, the site selection method, in addition to being useful for iterative optimization of binding specificity, can also be used as a useful indicator of binding affinity.
Example 4
Use of Site Selection to Identify Position-Dependent, GNN-Binding Zinc Fingers
A large number of engineered ZFPs have been evaluated, by site selection, to identify zinc fingers that bind to GNN target subsites. In the course of these studies, it became apparent that the binding specificity of a particular zinc finger sequence is, in some instances, dependent upon the position of the zinc finger in the protein, and hence upon the location of the target subsite within the target sequence. For example, if one wishes to design a three-finger zinc finger protein to bind to a target sequence containing the triplet subsite GAT, it is necessary to know whether this subsite is the first, second or third subsite in the target sequence (i.e., whether the GAT subsite will be bound by the first, second or third finger of the protein). Accordingly, over 110 three-finger zinc finger proteins, containing potential GNN-recognizing zinc fingers in various locations, have been evaluated by site selection experiments. Generally, several zinc finger sequences were designed to recognize each GNN triplet, and each design was tested in each of the F1, F2 and F3 positions through 4 to 6 rounds of selection.
The results of these analyses, shown in FIG. 2, provide optimal position-dependent zinc finger sequences (the sequences shown represent amino acid residues −1 through +6 of the recognition helix portion of the finger) for recognition of the 16 GNN target subsites, as well as site selection results for these GNN-specific zinc fingers. Optimal amino acid sequences for recognition of each GNN subsite from each of three positions (finger 1, finger 2 or finger 3) are thereby provided.
GNG-Binding Finger Designs
The amino acid sequence RSDXLXR (SEQ ID NO:4085) (position −1 to +6 of the recognition helix) was found to be optimal for binding to the four GNG triplets, with Asn+3 specifying A as the middle nucleotide; His+3 specifying G as the middle nucleotide; Ala+3 specifying T as the middle nucleotide; and Asp+3 specifying cytosine as the middle nucleotide. At the +5 position, Ala, Thr, Ser, and Gln, were tested, and all showed similar specificity profiles by site selection. Interestingly, and in contrast to a previous report (Swirnoff et al. (1995) Mol. Cell. Biol. 15:2275-2287), site selection results indicated that three naturally-occurring GCG-binding fingers from zif268 and Sp1, having the amino acid sequences RSDELTR (SEQ ID NO:123), RSDELQR (SEQ ID NO:302), and RSDERKR (SEQ ID NO:1100), were not GCG-specific. Rather, each of these fingers selected almost equal numbers of GCG and GTG sequences. Analysis of binding affinity by gel-shift experiments confirmed that finger 3 of zif268, having the sequence RSDERKR (SEQ ID NO:1000), binds GCG and GTG with approximately equal affinity.
Position Dependence of GCA-, GAT-, GGT-, GAA- and GCC-Binding Fingers
Based on existing design rules, the amino acid sequence QSGDLTR (SEQ ID NO:220) (−1 through +6) was tested for its ability to bind the GCA triplet from three positions (F1, F2, and F3) within a three-finger ZFP. FIG. 3A shows that the QSGDLTR (SEQ ID NO:220) sequence bound preferentially to the GCA triplet subsite from the F2 and F3 positions, but not from F1. In fact, the presence of QSGDLTR (SEQ ID NO:220) at the F1 position of three different three-finger ZFPs resulted predominantly in selection of GCT. Accordingly, an attempt was made to redesign this sequence to obtain specificity for GCA from the F1 position. Since the sequence Q−1G+2S+3R+6 (SEQ ID NO:4065) had previously been selected from a randomized F1 library using GCA as target (Rebar et al. (1994) Science 263:671-673), a D (asp) to S (ser) change was made at the +3 residue of this finger. The resulting sequence, QSGSLTR (SEQ ID NO:342), was tested for its binding specificity by site selection and found to preferentially bind GCA, from the F1 position, in three different ZFPs (see FIG. 2).
The QSGSLTR (SEQ ID NO:342) zinc finger, optimized for recognition of the GCA subsite from the F1 position, was tested for its selectivity when located at the F2 position. Accordingly, two ZFPs, one containing QSGSLTR (SEQ ID NO:342) at finger 2 and one containing QSGDLTR (SEQ ID NO:220) at finger 2 (both having identical F1 sequences and identical F3 sequences) were tested by site selection. The results indicated that, when used at the F2 position, QSGSLTR (SEQ ID NO:342) bound preferentially to GTA, rather than GCA. Thus, for optimal binding of a GCA triplet subsite from the F1 position, the amino acid sequence QSGSLTR (SEQ ID NO:342) is required; while, for optimal binding of the same subsite sequence from F2 or F3, QSGDLTR (SEQ ID NO:220) should be used. Accordingly, different zinc finger amino acid sequences may be needed to specify a particular triplet subsite sequence, depending upon the location of the subsite within the target sequence and, hence, upon the position of the finger in the protein.
Positional effects were also observed for zinc fingers recognizing GAT and GGT subsites. The zinc finger amino acid sequence QSSNLAR (SEQ ID NO:1765) (−1 through +6) is expected to bind to GAT, based on design rules. However, this sequence selected GAT only from the F1 position, and not from the F2 and F3 positions, from which the sequence GAA was preferentially bound (FIG. 3B). Similarly, the amino acid sequence QSSHLTR (SEQ ID NO:835) which, based on design rules, should bind GGT, selected GGT at the F1 position, but not at the F2 and F3 positions, from which it preferentially bound GGA (FIG. 3C). Conversely, the amino acid sequence TSGHLVR (SEQ ID NO:1425) has previously been disclosed to recognize the triplet GGT, based on its selection from a randomized library of zif268 finger 2. U.S. Pat. No. 6,140,081. However, TSGHLVR (SEQ ID NO:1425) was not specific for the GGT subsite when located at the F1 position (FIG. 3C). These results indicate that the binding specificity of many fingers is position dependent, and particularly point out that the sequence specificity of a zinc finger selected from a F2 library may be positionally limited.
The results shown in FIG. 2 indicate that recognition of at least GAA and GCC triplets by zinc fingers is also position dependent.
These positional dependences stand in contrast to earlier published work, which suggested that zinc fingers behaved as independent modules with respect to the sequence specificity of their binding to DNA. Desjarlais et al. (1993) Proc. Natl. Acad. Sci. USA 90:2256-2260.
Example 5
Characterization of EP2C
The engineered zinc finger protein EP2C binds to a target sequence, GCGGTGGCT with a dissociation constant (Kd) of 2 nM. Site selection results indicated that fingers 1 and 2 are highly specific for their target subsites, while finger 3 selects GCG (its intended target subsite) and GTG at approximately equal frequencies (FIG. 4A). To confirm these observations, the binding affinities of EP2C to its cognate target sequence, and to variant target sequences, was measured by standard gel-shift analyses (see Example 1, supra). As standards for comparison, the binding affinities of Sp1 and zif268 to their respective targets were also measured under the same conditions, and were determined to be 40 nM for SP1 (target sequence GGGGCGGGG) and 2 nM for zif268 (target sequence GCGTGGGCG). Measurements of binding affinities confirmed that F3 of EP2C bound GTG and GCG equally well (Kds of 2 nM), but bound GAG with a two-fold lower affinity (FIG. 4B). Finger 2 was very specific for the GTG triplet, binding 15-fold less tightly to a GGG triplet (compare 2C0 and 2C3 in FIG. 4B). Finger 1 was also very specific for the GCT triplet, it bound with 4-fold lower affinity to a GAT triplet (2C4) and with 2-fold lower affinity to a GCG triplet (2C5). This example shows, once again, the high degree of correlation between site selection results and binding affinities.
Example 6
Evaluation of Engineered ZFPs by In Vivo Functional Assays
To determine whether a correlation exists between the binding affinity of a engineered ZFP to its target sequence and its functionality in vivo, cell-based reporter gene assays were used to analyze the functional properties of the engineered ZFP EP2C (see Example 5, supra). For these assays, a plasmid encoding the EP2C ZFP, fused to a VP16 transcriptional activation domain, was used to construct a stable cell line (T-Rex-293TM, Invitrogen, Carlsbad, Calif.) in which expression of EP2C-VP16 is inducible, as described in Zhang et al., supra. To generate reporter constructs, three tandem copies of the EP2C target site, or its variants (see FIG. 4B, column 2), were inserted between the Mlu I and BglII sites of the pGL3 luciferase-encoding vector (Promega, Madison, Wis.), upstream of the SV40 promoter. Structures of all reporter constructs were confirmed by DNA sequencing.
Luciferase reporter assays were performed by co-transfection of luciferase reporter construct (200 ng) and pCMV-βgal (100 ng, used as an internal control) into the EP2C cells seeded in 6-well plates. Expression of the EP2C-VP16 transcriptional activator was induced with doxycycline (0.05 ug/ml) 24 h after transfection of reporter constructs. Cell lysates were harvested 40 hours post-transfection, luciferase and β-galactosidase activities were measured by the Dual-Light Reporter Assay System (Tropix, Bedford, Mass.), and luciferase activities were normalized to the co-transfected β-galactosidase activities. The results, shown on the right side of FIG. 4B, showed that the normalized luciferase activity for each reporter construct was well correlated with the in vitro binding affinity of EP2C to the target sequence present in the construct. For example, the target sequences to which EP2C bound with greatest affinity (2C0 and 2C2, Kd of 2 nM for each) both stimulated the highest levels of luciferase activity, when used to drive luciferase expression in the reporter construct (FIG. 4B). Target sequences to which EP2C bound with 2-fold lower affinity, 2C1 and 2C5 (Kd of 4 nM for each), stimulated roughly half the luciferase activity of the 2C0 and 2C2 targets. The 2C3 and 2C4 sequences, for which EP2C showed the lowest in vitro binding affinities, also yielded the lowest levels of in vivo activity when used to drive luciferase expression. Target 3B, a sequence to which EP2C does not bind, yielded background levels of luciferase activity, similar to those obtained with a luciferase-encoding vector lacking EP2C target sequences (pGL3). Thus there exist good correlations between binding affinity (as determined by Kd measurement), binding specificity (as determined by site selection) and in vivo functionality for engineered zinc finger proteins.

















TABLE 1







SEQ

SEQ

SEQ

SEQ
Kd



SBS#
TARGET
ID
F1
ID
F2
ID
F3
ID
(nM)

























249
GCGGGGGCG
17
RSDELTR
123
RSDHLSR
229
RSDELRR
335
20



 


250
GCGGGGGCG
18
RSDELTR
124
RSDHLSR
230
RSDTLKK
336
70


 


251
GCGGAGGCG
19
RSDELTR
125
RSDNLTR
231
RSDELRR
337
27.5


 


252
GCGGCCGCG
20
RSDELTR
126
DRSSLTR
232
RSDELRR
338
100


 


253
GGATGGGGG
21
RSDHLAR
127
RSDHLTT
233
QPAHLAR
339
0.75


 


256
GCGGGGTCC
22
ERGDLTT
128
RSDHLSR
234
RSDELRR
340
800


 


258
GCGGGCGGG
23
RSDHLTR
129
ERGHLTR
235
RSDELRR
341
15


 


259
GCAGAGGAG
24
RSDNLAR
130
RSDNLAR
236
QSGSLTR
342
250


 


261
GAGGTGGCC
25
ERGTLAR
131
RSDALSR
237
RSDNLSR
343
0.5


 


262
GCGGGGGCT
26
QSSDLQR
132
RSDHLSR
238
RSDELRR
344
20


 


263
GCGGGGGCT
27
QSSDLQR
133
RSDHLSR
239
RSDTLKK
345
1


 


264
GTGGCTGCC
28
DRSSLTR
134
QSSDLQR
240
RSDALAR
346
27


 


265
GTGGCTGCC
29
ERGTLAR
135
QSSDLQR
241
RSDALAR
347
600


 


269
GGGGCCGGG
30
RSDHLTR
136
DRSSLTR
242
RSDHLTR
348
5


 


270
GGGGCCGGG
31
RSDHLTR
137
ERGTLAR
243
RSDHLTR
349
52.5


 


272
GCAGGGGCC
32
DRSSLTR
138
RSDHLSR
244
QSGSLTR
350
20


 


337
TGCGGGGCAA
33
RSADLTR
139
RSDHLTR
245
ERQHLAT
351
24


 


338
TGCGGGGCAA
34
RSADLTR
140
RSDHLTR
246
ERDHLRT
352
8


 


339
TGCGGGGCAA
35
RSADLTR
141
RSDHLTT
247
ERQHLAT
353
64


 


340
TGCGGGGCAA
36
RSADLTR
142
RSDHLTT
248
ERDHLRT
354
48


 


341
TGCGGGGCAA
37
RSADLTR
143
RGDHLKD
249
ERQHLAT
355
1000


 


342
TGCGGGGCAA
38
RSADLTR
144
RGDHLKD
250
ERDHLRT
356
1000


 


343
TGCGGGGCAA
39
QSGSLTR
145
RSDHLTR
251
ERQHLAT
357
8


 


344
TGCGGGGCAA
40
QSGSLTR
146
RSDHLTR
252
ERDHLRT
358
6


 


345
TGCGGGGCAA
41
QSGSLTR
147
RSDHLTT
253
ERQHLAT
359
96


 


346
TGCGGGGCAA
42
QSGSLTR
148
RSDHLTT
254
ERDHLRT
360
64


 


347
TGCGGGGCAA
43
QSGSLTR
149
RGDHLKD
255
ERQHLAT
361
1000


 


348
TGCGGGGCAA
44
QSGSLTR
150
RGDHLKD
256
ERDHLRT
362
1000


 


367
GGGGGCGGG
45
RSDHLTR
151
DSGHLTR
257
RSDHLQR
363
60


 


368
GAGGGGGCG
46
RSDELTR
152
RSDHLTR
258
RSDNLTR
364
3.5


 


369
GTAGTTGTG
47
RSDALTR
153
TGGSLAR
259
QSGSLTR
365
95


 


370
GTAGTTGTG
48
RSDALTR
154
NRATLAR
260
QSASLTR
366
300


 


371
GTAGTTGTG
49
RSDALTR
155
NRATLAR
261
QSGSLTR
367
175


 


372
GTAGTTGTG
50
RSDSLLR
156
TGGSLAR
262
QSASLTR
368
112.5


 


373
GTAGTTGTG
51
RSDSLLR
157
NPATLAR
263
QSASLTR
369
320


 


374
GCTGAGGAA
52
QRSNLVR
158
RSDNLTR
264
TSSELQR
370
3.3


 


375
GAGGAAGAT
53
QQSNLAR
159
QSGNLQR
265
RSDNLTR
371
85


 


401
GTAGTTGTG
54
RSDALTR
160
TGGSLAR
266
QSASLTR
372
80


 


403
GTAGTTGTG
55
RSDSLLR
161
NRATLAR
267
QSGSLTR
373
750


 


421
GTAGTTGTG
56
DSDSLLR
162
TGGSLAR
268
QSGSLTR
374
500


 


422
GTAGTTGTG
57
RSDSLLR
163
TGGSLTR
269
QSGSLTR
375
200


 


423
GTAGTTGTG
58
RSDALTR
164
TGGSLAR
270
QRSALAR
376
1000


 


424
GATGCTGAG
59
RSDNLTR
165
TSSELQR
271
TSANLSR
377
100


 


425
GATGCTGAG
60
RSDNLTR
166
QSSDLQR
272
QQSNLAR
378
25


 


426
GATGCTGAG
61
RSDNLTR
167
QSSDLQR
273
TSANLSR
379
5.5


 


427
GCTGAGGAA
62
QRSNLVR
168
RSDNLTR
274
QSSDLQR
380
1


 


428
GAAGATGAC
63
DSSNLTR
169
QQSNLAR
275
QRSNLVR
381
120


 


429
GAAGATGAC
64
DSSNLTR
170
TSANLSR
276
QRSNLVR
382
50


 


430
GATGACGAC
65
EKANLTR
171
DSSNLTR
277
QQSNLAR
383
250


 


431
GACGACGGC
66
DSGHLTR
172
DRSNLER
278
DSSNLTR
384
100


 


432
GACGACGGC
67
DSGHLTR
173
DHANLAR
279
DSSNLTR
385
1000


 


433
GACGACGGC
68
DSGNLTR
174
DHANLAR
280
DSSNLTR
386
1000


 


434
GACGGCGTA
69
QSASLTR
175
DSGHLTR
281
EKANLTR
387
152.5


 


435
GACGGCGTA
70
QSASLTR
176
DSGHLTR
282
ERGNLTR
388
150


 


436
GACGGCGTA
71
QRSALAR
177
DSGHLTR
283
EKANLTR
389
95


 


437
GACGGCGTA
72
QRSALAR
178
DSGHLTR
284
ERGNLTR
390
117.5


 


438
GAGGGGGCG
73
RSDELTR
179
RSDHLTT
285
RSDNLTR
391
62.5


 


440
GCCGAGGTGC
74
RSDSLLR
180
RSKNLQR
286
ERGTLAR
392
40


 


441
GGTGGAGTCA
75
DSGSLTR
181
QSGHLQR
287
TSGHLTR
393
250


 


445
GTCGCAGTGA
76
RSDSLRR
182
QSSDLQK
288
DSGSLTR
394
1000


 


450
GACTTGGTGC
77
RSDTLAR
183
RGDALTS
289
DRSNLTR
395
130


 


453
GGTGGAGTCA
78
DRSALAR
184
QSGHLQR
290
DSSKLSR
396
150


 


461
GAGTACTGTA
79
QRSHLTT
185
DRSNLRT
291
RSDNLAR
397
120


 


463
GTGGAGGAGA
80
RSDNLTR
186
RSDNLAR
292
RSDALAR
398
0.5


 


464
GTGGAGGAGA
81
RSDNLTR
187
RSDNLAR
293
RSDSLAR
399
0.4


 


466
CAGGCTGCGC
82
RSDDLTR
188
QSSDLQR
294
RSDNLRE
400
65


 


467
CAGGCTGCGC
83
RSDELTR
189
QSSDLQR
295
RGDHLKD
401
800


 


468
CAGGCTGCGC
84
RSDDLTR
190
QSSDLQR
296
RGDHLKD
402
42


 


469
GAAGAGGTCT
85
DRSALAR
191
RSDNLAR
297
QSGNLTR
403
13.5


 


472
GAGGTCTGGA
86
RSSHLTT
192
DRSALAR
298
RSDNLAR
404
80


 


476
GGAGAGGATG
87
TTSNLRR
193
RSDNLAR
299
QSDHLTR
405
80


 


477
GGAGAGGATG
88
TTSNLRR
194
RSDNLAR
300
QRAHLAR
406
100


 


478
GGAGAGGATG
89
TTSNLRR
195
RSDNLAR
301
QSGHLRR
407
60


 


479
GTGGCGGACC
90
DSSNLTR
196
RSDELQR
302
RSDALAR
408
8.5


 


480
GTGGCGGACC
91
DSSNLTR
197
RADTLRR
303
RSDALAR
409
5


 


483
GAGGGCGAAG
92
QSANLAR
198
ESSKLKR
304
RSDNLAR
410
130


 


484
GAGGGCGAAG
93
QSDNLAR
199
ESSKLKR
305
RSDNLAR
411
1000


 


485
GGAGAGGTTT
94
QSSALAR
200
RSDNLAR
306
QRAHLAR
412
110


 


487
GGAGAGGTTT
95
NRATLAR
201
RSDNLAR
307
QSGHLAR
413
76.9


 


488
TGGTAGGGGG
96
RSDHLAR
202
RSDNLTT
308
RSDHLTT
414
35


 


490
TAGGGGGTGG
97
RSDSLLR
203
RSDHLTR
309
RSDNLTT
415
1.5


 


503
GCCGAGGTGC
98
RSDSLLR
204
RSDNLAR
310
ERGTLAR
416
50


 


504
GCCGAGGTGC
99
RSDSLLR
205
RSDNLAR
311
DRSDLTR
417
25


 


505
GCCGAGGTGC
100
RSDSLLR
206
RSDNLAR
312
DCRDLAR
418
65


 


526
GCGGGCGGGC
101
RSDHLTR
207
ERGHLTR
313
RSDTLKK
419
8


 


543
GAGTGTGTGA
102
RSDLLQR
208
MSHHLKE
314
RSDHLSR
420
50


 


544
GAGTGTGTGA
103
RSDSLLR
209
MSHHLKE
315
RSDNLAR
421
125


 


545
GAGTGTGTGA
104
RKDSLVR
210
TSDHLAS
316
RSDNLTR
422
32


 


546
GAGTGTGTGA
105
RSDLLQR
211
MSHHLKT
317
RLDGLRT
423
500


 


547
GAGTGTGTGA
106
RKDSLVR
212
TSGHLTS
318
RSDNLTR
424
500


 


548
GAGTGTGTGA
107
RSSLLQR
213
MSHHLKT
319
RSDHLSR
425
500


 


549
GAGTGTGTGA
108
RSSLLQR
214
MSHHLKE
320
RSDHLSR
426
500


 


550
GAGTGTGTGA
109
RKDSLVR
215
TKDHLAS
321
RSDNLTR
427
20


 


551
GAGTGTGTGA
110
RSDLLQR
216
MSHHLKT
322
RSDHLSR
428
50


 


552
GAGTGTGTGA
111
RKDSLVR
217
MSHHLKT
323
RSDNLTR
429
31


 


553
GAGTGTGTGA
112
RSDSLLR
218
MSHHLKE
324
RSDNLTR
430
125


 


554
GAGTGTGTGA
113
RKDSLVR
219
TSDHLAS
325
RSDNLAR
431
62.5


 


558
TGCGGGGCA
114
QSGDLTR
220
RSDHLTR
326
DSGHLAS
432
21


 


559
GAGTGTGTGA
115
RSDSLLR
221
TSDHLAS
327
RSDNLAR
433
1000


 


560
GAGTGTGTGA
116
RSSLLQR
222
MSHHLKT
328
RSDHLSR
434
500


 


561
GAGTGTGTGA
117
RKDSLVR
223
MSHHLKE
329
RSDNLAR
435
1000


 


562
GAGTGTGTGA
118
RSDSLLR
224
TSGHLTS
330
RSDNLAR
436
1000


 


565
GATGCTGAG
119
RSDNLTR
225
TSSELQR
331
QQSNLAR
437
100


 


567
GAAGATGAC
120
EKANLTR
226
TSANLSR
332
QRSNLVR
438
47.5


 


568
GATGACGAC
121
EKAIThTR
227
DSSNLTR
333
TSANLSR
439
300


 


569
GTAGTTGTG
122
RSDSLLR
228
TGGSLAR
334
QRSALTR
440
52


























TABLE 2







SEQ

SEQ

SEQ

SEQ
Kd



SBS#
TARGET
ID
F1
ID
F2
ID
F3
ID
(nM)

























201
GCAGCCTTG
441
RSDSLTS
646
ERSTLTR
851
QRADLRR
1056
1000



 


202
GCAGCCTTG
442
RSDSLTS
647
ERSTLTR
852
QRADLAR
1057
1000


 


203
GCAGCCTTG
443
RSDSLTS
648
ERSTLTR
853
QPATLRR
1058
1000


 


204
GCAGCCTTG
444
RSDSLTS
649
ERSTLTR
854
QRATLAR
1059
1000


 


205
GAGGTAGAA
445
QSANLAR
650
QSATLAR
855
RSDNLSR
1060
80


 


206
GAGGTAGAA
446
QSANLAR
651
QSAVLAR
856
RSDNLSR
1061
1000


 


207
GAGTGGTTA
447
QRASLAS
652
RSDHLTT
857
RSDNLAR
1062
70


 


208
TAGGTCTTA
448
QRASLAS
653
DRSALAR
858
RSDNLAS
1063
1000


 


209
GGAGTGGTT
449
QSSALAR
654
RSDALAR
859
QRAHLAR
1064
35


 


210
GGAGTGGTT
450
NRDTLAR
655
RSDALAR
860
QPAHLAR
1065
65


 


211
GGAGTGGTT
451
QSSALAR
656
RSDALAS
861
QRAHLAR
1066
140


 


212
GGAGTGGTT
452
NRDTLAR
657
RSDALAS
862
QRAHLAR
1067
400


 


213
GTTGCTGGA
453
QRAHLAR
658
QSSTLAR
863
QSSALAR
1068
1000


 


214
GTTGCTGGA
454
QRAHLAR
659
QSSTLAR
864
NRDTLAR
1069
1000


 


215
GAAGTCTGT
455
NRDHLMV
660
DRSALAR
865
QSANLSR
1070
1000


 


216
GAAGTCTGT
456
NRDHLTT
661
DRSALAR
866
QSANLSR
1071
1000


 


217
GAGGTCGTA
457
QRSALAR
662
DRSALAR
867
RSDNLAR
1072
40


 


219
GATGTTGAT
458
QQSNLAR
663
NRDTLAR
868
NRDNLSR
1073
1000


 


220
GATGTTGAT
459
QQSNLAR
664
NRDTLAR
869
QQSNLSR
1074
1000


 


221
GATGAGTAC
460
DRSNLRT
665
RSDNLAR
870
NRDNLAR
1075
1000


 


222
GATGAGTAC
461
ERSNLRT
666
RSDNLAR
871
NRDNLAR
1076
1000


 


223
GATGAGTAC
462
DRSNLRT
667
RSDNLAR
872
QQSNLAR
1077
105


 


224
GATGAGTAC
463
ERSNLRT
668
RSDNLAR
873
QQSNLAR
1078
1000


 


225
TGGGAGGTC
464
DRSALAR
669
RSDNLAR
874
RSDHLTT
1079
6


 


226
GCAGCCTTG
465
RGDALTS
670
ERGTLAR
875
QSGSLTR
1080
1000


 


227
GCAGCCTTG
466
RGDALTV
671
ERGTLAR
876
QSGSLTR
1081
1000


 


228
GCAGCCTTG
467
RGDALTM
672
ERGTLAR
877
QSGSLTR
1082
1000


 


229
GCAGCCTTG
468
RGDALTS
673
ERGTLAR
878
RSDELTR
1083
1000


 


230
GCAGCCTTG
469
RGDALTV
674
ERGTLAR
879
RSDELTR
1084
1000


 


231
GCAGCCTTG
470
RGDALTM
675
ERGTLAR
880
RSDELTR
1085
1000


 


232
GGTGTGGTG
471
RSDALTR
676
RSDALAR
881
NRSHLAR
1086
50


 


233
GGTGTGGTG
472
RSDALTR
677
RSDALAR
882
QASHLAR
1087
100


 


235
GTAGAGGTG
473
RSDALTR
678
RSDNLAR
883
QRGALAR
1088
80


 


236
GGGGAGGGG
474
RSDHLAR
679
RSDNLAR
884
RSDHLSR
1089
0.3


 


237
GGGGAGGCC
475
ERGTLAR
680
RSDNLAR
885
RSDHLSR
1090
0.3


 


238
GGGGAGGCC
476
ERGTLAR
681
RSDNLQR
886
RSDHLSR
1091
0.8


 


239
GGCGGGGAG
477
RSDNLTR
682
RSDHLTR
887
DRSHLAR
1092
0.4


 


240
GCAGGGGAG
478
RSDNLTR
683
RSDHLSR
888
QSGSLTR
1093
1


 


242
GGGGGTGCT
479
QSSDLRR
684
QSSHLAR
889
RSDHLSR
1094
1


 


243
GTGGGCGCT
480
QSSDLRR
685
DRSHLAR
890
RSDALAR
1095
75


 


244
TAAGAAGGG
481
RSDHLAR
686
QSGNLTR
891
QSGNLRT
1096
100


 


245
TAAGAAGGG
482
RSDHLAR
687
QSANLTR
892
QSGNLRT
1097
235


 


246
GAAGGGGAG
483
RSDNLAR
688
RSDHLAR
893
QSGNLTR
1098
2


 


247
GAAGGGGAG
484
RSDNLAR
689
RSDHLAR
894
QSGNLRR
1099
2


 


276
GCGGCCGCG
485
RSDELTR
690
ERGTLAR
895
RSDERKR
1100
90


 


277
GCGGCCGCG
486
RSDELTR
691
DRSSLTR
896
RSDERKR
1101
107


 


278
GCGGCCGCG
487
QSWELTR
692
ERGTLAR
897
RSDERKR
1102
190


 


279
GCGGCCGCG
488
QSWELTR
693
DRSSLTR
898
RSDERKR
1103
260


 


280
GCGGCCGCG
489
QSGSLTR
694
ERGTLAR
899
RSDERKR
1104
160


 


281
GCGGCCGCG
490
QSGSLTR
695
DRSSLTR
900
RSDERKR
1105
225


 


282
GCAGAAGTG
491
RGDALTR
696
QSANLTR
901
QSADLAR
1106
1000


 


283
GCAGAAGTG
492
RSDALTR
697
QSGNLTR
902
QSGSLTR
1107
2


 


284
GCGGCCGCG
493
QSGSLTR
698
RSDHLTT
903
RSDERKR
1108
1000


 


285
TGTGCGGCC
494
ERGTLAR
699
RSDELTR
904
SRDHLQS
1109
1000


 


287
GCAGAAGCG
495
RGPDLAR
700
QSANLTR
905
QSGSLTR
1110
1000


 


288
GCAGAAGCG
496
RGPDLAR
701
QSANLTR
906
QSGSLTR
1111
1000


 


289
GCAGAAGCG
497
RGPDLAR
702
QSGNLQR
907
QSGSLTR
1112
800


 


290
GCAGAAGCG
498
RSDELAR
703
QSANLQR
908
QSADLAR
1113
1000


 


292
GCAGAAGCG
499
RSDELTR
704
QSANLQR
909
QSGSLTR
1114
1000


 


293
GTGTGCGGC
500
DRSHLTR
705
ERHSLQT
910
RSDALTR
1115
320


 


296
TGCGCGGCC
501
ERGTLAR
706
RSDELTR
911
DRDHLQS
1116
1000


 


297
TGCGCGGCC
502
ERGTLAR
707
RSDELRR
912
DRSHLQT
1117
500


 


298
GCTTAGGCA
503
QTGELRR
708
RSDNLQK
913
TSGDLSR
1118
4000


 


299
GCTTAGGCA
504
QTSDLRR
709
RSDNLQK
914
QSSDLQR
1119
4000


 


300
GCTTAGGCA
505
QTADLRR
710
RSDNLQR
915
QSSDLSR
1120
400


 


301
GCTTAGGCA
506
QSADLRR
711
RSDNLQT
916
QSSDLSR
1121
350


 


302
GCTTAGGCA
507
QSGSLTR
712
RSDNLQT
917
QSSDLSR
1122
75


 


303
GCTTAGGCA
508
QTGSLTR
713
RSDNLQT
918
QSSDLSR
1123
135


 


304
GCTTAGGCA
509
QTADLTR
714
RSDNLQT
919
QSSDLSR
1124
230


 


305
GCTTAGGCA
510
QTGDLTR
715
RSDNLQT
920
QSSDLSR
1125
230


 


306
GCTTAGGCA
511
QTASLTR
716
RSDNLQT
921
QSSDLSR
1126
280


 


307
GAAGAAGCG
512
RSDELRR
717
QSGNLQR
922
QSGNLSR
1127
50.5


 


308
GAAGAAGCG
513
RSDELRR
718
QSANLQR
923
QSANLQR
1128
1000


 


309
GGAGATGCC
514
ERSDLRR
719
QSSNLQR
924
QSGHLSR
1129
4000


 


310
GGAGATGCC
515
DRSDLTR
720
NRDNLQT
925
QSGHLSR
1130
1000


 


311
GGAGATGCC
516
DRSTLTR
721
NRDNLQR
926
QSGHLSR
1131
170


 


312
GGAGATGCC
517
ERGTLAR
722
NRDNLQR
927
QSGHLSR
1132
2000


 


313
GGAGATGCC
518
DRSDLTR
723
QRSNLQR
928
QSGHLSR
1133
1000


 


314
GGAGATGCC
519
DRSSLTR
724
QSSNLQR
929
QSGHLSR
1134
117.5


 


315
GGAGATGCC
520
ERGTLAR
725
QSSNLQR
930
QSGHLSR
1135
265


 


316
GGAGATGCC
521
ERGTLAR
726
QRDNLQR
931
QSGHLSR
1136
3000


 


318
TAGGAGATGC
522
RSDALTS
727
RSDNLAR
932
RSDNLAS
1137
100


 


319
GGGGAAGGG
523
KTSHLRA
728
QSGNLSR
933
RSDHLSR
1138
125


 


320
GGGGAAGGG
524
RSDHLTR
729
QSGNLSR
934
RSDHLSR
1139
5


 


321
GGCGGAGAT
525
TTSNLRR
730
QSGHLQR
935
DRSHLTR
1140
200


 


323
GGCGGAGAT
526
TTSNLRR
731
QSGHLQR
936
DRDHLTR
1141
600


 


324
GGCGGAGAT
527
TTSNLRR
732
QSGHLQR
937
DRDHLTR
1142
200


 


325
GTATCTGCT
528
NSSDLTR
733
NSDVLTS
938
QSDVLTR
1143
1000


 


326
GTATCTGTT
529
NSDALTR
734
NSDVLTS
939
QSDVLTR
1144
1000


 


327
TCTGCTGGG
530
RSDHLTR
735
NSADLTR
940
NSDDLTR
1145
1000


 


328
TCTGTTGGG
531
RSDHLTR
736
NSSALTS
941
NSDDLTR
1146
1000


 


349
GGTGTCGCC
532
DCRDLAR
737
DSGSLTR
942
TSGHLTR
1147
1000


 


350
TCCGAGGGT
533
TSGHLTR
738
RSDNLTR
943
DCRDLTT
1148
332


 


351
GCTGGTGTC
534
DSGSLTR
739
TSGHLTR
944
TLHTLTR
1149
1000


 


352
GGAGGGGTG
535
RSDSLLR
740
RSDHLTR
945
QSDHLTR
1150
26


 


353
GTTGGAGCC
536
DCRDLAR
741
QSDHLTR
946
TSGALTR
1151
1000


 


354
GAAGAGGAC
537
DSSNLTR
742
RSDNLTR
947
QRSNLVR
1152
28


 


355
GAAGAGGAC
538
EKANLTR
743
RSDNLTR
948
QRSNLVR
1153
20


 


356
GGCTGGGCG
539
RSDELRR
744
RSDHLTK
949
DSDHLSR
1154
1000


 


357
GGCTGGGCG
540
RSDELRR
745
RSDHLTK
950
DSDHLSR
1155
1000


 


358
GGCTGGGCG
541
RSDELRR
746
RSDHLTK
951
DSSHLSR
1156
225


 


361
GGGTTTGGG
542
RSDHLTR
747
QSSALTR
952
RSDHLTR
1157
130


 


363
GGGTTTGGG
543
RSDHLTR
748
QSSVLTR
953
RSDHLTR
1158
200


 


364
GTGTCCGAAG
544
RSDNLTR
749
DSAVLTT
954
RSDSLTR
1159
1000


 


365
GGTGCTGGT
545
QASHLTR
750
QASVLTR
955
QASHLTR
1160
600


 


366
GAGGGTGCT
546
QASVLTR
751
QASHLTR
956
RSDNLTR
1161
1000


 


367
GGGGGCGGG
547
RSDHLTR
752
DSGHLTR
957
RSDHLQR
1162
60


 


368
GAGGGGGCG
548
RSDELTR
753
RSDHLTR
958
RSDNLTR
1163
3.5


 


369
GTAGTTGTG
549
RSDALTR
754
TGGSLAR
959
QSGSLTR
1164
95


 


370
GTAGTTGTG
550
RSDALTR
755
NRATLAR
960
QSASLTR
1165
300


 


371
GTAGTTGTG
551
RSDALTR
756
NRATLAR
961
QSGSLTR
1166
175


 


372
GTAGTTGTG
552
RSDSLLR
757
TGGSLAR
962
QSASLTR
1167
112.5


 


373
GTAGTTGTG
553
RSDSLLR
758
NRATLAR
963
QSASLTR
1168
320


 


374
GCTGAGGAA
554
QRSNLVR
759
RSDNLTR
964
TSSELQR
1169
3.3


 


375
GAGGAAGAT
555
QQSNLAR
760
QSGNLQR
965
RSDNLTR
1170
85


 


377
GTGTTGGCAG
556
QSGSLTR
761
RGDALTS
966
RSDALTR
1171
89


 


378
GCCGAGGAGA
557
RSDNLTR
762
RSDNLTR
967
DRSSLTR
1172
31


 


379
GCCGAGGAGA
558
RSDNLTR
763
RSDNLTR
968
ERGTLAR
1173
3


 


380
GAGTCGGAAG
559
QSANLAR
764
RSDELTT
969
RSDNLAR
1174
1000


 


381
GCAGCTGCGC
560
RSDELTR
765
QSSDLQR
970
QSGDLTR
1175
1.5


 


383
TGGTTGGTAT
561
QSATLAR
766
RGDALTS
971
RSDHLTT
1176
1000


 


384
GTGGGCTTCA
562
DRSALTT
767
DRSHLAR
972
RSDALAR
1177
60


 


385
GGGGCGGAGC
563
RSDNLTR
768
RSDTLKK
973
RSDHLSR
1178
1.2


 


386
GGGGCGGAGC
564
RSDNLTR
769
RSDELQR
974
RSDHLSR
1179
0.4


 


387
GGCGAGGCAA
565
QSGSLTR
770
RSDNLAR
975
DRSHLAR
1180
2.5


 


388
GGCGAGGCAA
566
QSGDLTR
771
RSDNLAR
976
DRSHLAR
1181
28


 


390
GTGGCAGCGG
567
RSDTLKK
772
QSSDLQK
977
RSDALAR
1182
20


 


392
GTGGCAGCGG
568
RSDELTR
773
QSSDLQK
978
RSDALAR
1183
1000


 


396
GCGGGAGCAG
569
QSGSLTR
774
QSGHLQR
979
RSDTLKK
1184
18.8


 


397
GCGGGAGCAG
570
QSGDLTR
775
QSGHLQR
980
RSDTLKK
1185
25


 


400
TCAGTGGTGG
571
RSDALAR
776
RSDSLAR
981
QSGDLRT
1186
40


 


405
GCGGCCGCA
572
RSDELTR
777
ERGTLAR
982
RSDERKR
1187
110


 


406
GCGGCCGCA
573
RSDELTR
778
DRSSLTR
983
RSDERKR
1188
110


 


407
GCGGCCGCA
574
QSWELTR
779
ERGTLAR
984
RSDERKR
1189
410


 


408
GCGGCCGCA
575
QSWELTR
780
DRSSLTR
985
RSDERKR
1190
380


 


409
GCGGCCGCA
576
QSGSLTR
781
ERGTLAR
986
RSDERKR
1191
50


 


410
GCAGAAGTC
577
RSDALTR
782
QSGNLTR
987
QSGSLTR
1192
3


 


411
GCGGCCGCA
578
QSGSLTR
783
RSDHLTT
988
RSDERKR
1193
1000


 


412
GCGTGGGCG
579
QSGSLTR
784
RSDHLTT
989
RSDERKR
1194
5


 


413
GCGTGGGCA
580
QSGSLTR
785
RSDHLTT
990
RSDERKR
1195
5


 


414
GCAGAAGCA
581
RSDELTR
786
QSANLQR
991
QSGSLTR
1196
1000


 


415
GTGTGCGGA
582
DRSHLTR
787
ERHSLQT
992
RSDALTR
1197
1000


 


416
TGTGCGGCC
583
ERGTLAR
788
RSDELRR
993
DRSHLQT
1198
1000


 


493
GGGGTGGCGG
584
RSDTLKK
789
RSDSLAR
994
RSDHLSR
1199
300


 


494
GCCGAGGAGA
585
RSDNLTR
790
RSDNLTR
995
DRSSLTR
1200
90


 


496
GGTGGTGGC
586
DTSHLRR
791
TSGHLQR
996
TSGHLSR
1201
1000


 


497
GTTTGCGTC
587
ETASLRR
792
DSAHLQR
997
TSSALSR
1202
1000


 


498
GAAGAGGCA
588
QTGELRR
793
RSDNLQR
998
QSGNLSR
1203
30


 


499
GCTTGTGAG
589
RTSNLRR
794
TSSHLQK
999
DTDHLRR
1204
1000


 


500
GCTTGTGAG
590
RSDNLTR
795
QSSNLQT
1000
DRSHLAR
1205
1000


 


501
GTGGGGGTT
591
NRATLAR
796
RSDHLSR
1001
RSDALAR
1206
8


 


502
GGGGTGGGA
592
QSAHLAR
797
RSDALAR
1002
RSDHLSR
1207
60


 


507
GAGGTAGAGG
593
RSDNLAR
798
QRSALAR
1003
RSDNLAR
1208
10


 


508
GAGGTAGAGG
594
RSDNLAR
799
QSATLAR
1004
RSDNLAR
1209
10


 


509
GTCGTGTGGC
595
RSDHLTT
800
RSDALAR
1005
DRSALAR
1210
100


 


510
GTTGAGGAAG
596
QSGNLAR
801
RSDNLAR
1006
NPATLAR
1211
100


 


511
GTTGAGGAAG
597
QSGNLAR
802
RSDNLAR
1007
QSSALAR
1212
100


 


512
GAGGTGGAAG
598
QSGNLAR
803
RSDALAR
1008
RSDNLAR
1213
10


 


513
GAGGTGGAAG
599
QSANLAR
804
RSDALAR
1009
RSDNLAR
1214
1.5


 


514
TAGGTGGTGG
600
RSDALTR
805
RSDALAR
1010
RSDNLTT
1215
10


 


515
TGGGAGGAGT
601
RSDNLTR
806
RSDNLTR
1011
RSDHLTT
1216
0.5


 


516
GGAGGAGCT
602
TTSELRR
807
QSGHLQR
1012
QSGHLSR
1217
700


 


517
GGAGCTGGGG
603
RTDHLRR
808
TSSELQR
1013
QSGHLSR
1218
50


 


518
GGGGGAGGAG
604
QTGHLRR
809
QSGHLQR
1014
RSDHLSR
1219
30


 


519
GGGGAGGAGA
605
RSDNLAR
810
RSDNLSR
1015
RSDHLSR
1220
0.3


 


520
GGAGGAGAT
606
TTANLRR
811
QSGHLQR
1016
QSGHLSR
1221
300


 


521
GCAGCAGGA
607
QTGHLRR
812
QSGELQR
1017
QSGELSR
1222
1000


 


522
GATGAGGCA
608
QTGELRR
813
RSDNLQR
1018
TSANLSR
1223
200


 


527
GGGGAGGATC
609
TTSNLRR
814
RSSNLQR
1019
RSDHLSR
1224
2


 


528
GGGGAGGATC
610
TTSNLRR
815
RSSNLQR
1020
RSDHLSR
1225
10


 


529
GAGGCTTGGG
611
RTDHLRK
816
TSAELQR
1021
RSSNLSR
1226
1000


 


531
GCGGAGGCTT
612
TTGELRR
817
RSSNLQR
1022
RSDELSR
1227
160


 


532
GCGGAGGCTT
613
QSSDLQR
818
RSSNLQR
1023
RSDELSR
1228
100


 


533
GCGGAGGCTT
614
QSSDLQR
819
RSDNLAR
1024
RSADLSR
1229
7


 


534
GCGGAGGCTT
615
QSSDLQR
820
RSDNLAR
1025
RSDDLRR
1230
10


 


535
GCAGCCGGG
616
RTDHLRR
821
ESSDLQR
1026
QSGELSR
1231
1000


 


538
GCAGAGGCTT
617
QSSDLQR
822
RSDNLAR
1027
QSGSLTR
1232
70


 


540
TGGGCAGGCC
618
DRSHLTR
823
QSGSLTR
1028
RSDHLTT
1233
55


 


541
GGGGAGGAT
619
TTSNLRR
824
RSSNLQR
1029
RSDHLSR
1234
3


 


570
GGGGAAGGCT
620
DSGHLTR
825
QRSNLVR
1030
RSDHLTR
1235
20


 


571
GTGTGTGTGT
621
RSDSLTR
826
QRSNLVR
1031
RSDSLLR
1236
1000


 


572
GCATACGTGG
622
RSDSLLR
827
DKGNLQS
1032
QSDDLTR
1237
1000


 


573
GCATACGTG
623
RSDSLLR
828
DKGNLQS
1033
QSGDLTR
1238
1000


 


574
TACGTGGGGT
624
RSDHLTR
829
RSDHLTR
1034
DKGNLQT
1239
25


 


575
TACGTGGGCT
625
DFSHLTR
830
RSDHLTR
1035
DKGNLQT
1240
472


 


576
GAGGGTGTTG
626
NSDTLAR
831
TSGHLTR
1036
RSDNLTR
1241
200


 


577
GGAGCGGGGA
627
RSDHLSR
832
RSDELQR
1037
QSDHLTR
1242
200


 


579
GGGGTTGAGG
628
RSDNLTR
833
NRDTLAR
1038
TSGHLTR
1243
200


 


580
GGTGTTGGAG
629
QPAHLAR
834
NRDTLAR
1039
TSGHLTR
1244
1000


 


581
TACGTGGGTT
630
QSSHLTR
835
RSDSLLR
1040
DKGNLQT
1245
382


 


583
GTAGGGGTTG
631
NSSALTR
836
RSDHLTR
1041
QSASLTR
1246
46


 


584
GAAGGCGGAG
632
QAGHLTR
837
DKSHLTR
1042
QSGNLTR
1247
1000


 


585
GAAGGCGGAG
633
QAGHLTR
838
DSGHLTR
1043
QSGNLTR
1248
1000


 


587
GGGGGTTACG
634
DKGNLQT
839
TSGHLTR
1044
RSDHLSK
1249
500


 


588
GGGGGGGGGG
635
RSDHLSR
840
RSDHLTR
1045
RSDHLSK
1250
30


 


589
GGAGTATGCT
636
DSGHLAS
841
QSATLAR
1046
QSDHLTR
1251
1000


 


595
TGGTTGGTAT
637
QRGSLAR
842
RGDALTR
1047
RSDHLTT
1252
73.3


 


597
TGGTTGGTA
638
QNSAMRK
843
RGDALTS
1048
RSDHLTT
1253
1000


 


598
TGGTTGGTA
639
QRGSLAR
844
RDGSLTS
1049
RSDHLTT
1254
1000


 


599
TGGTTGGTA
640
QNSAMRK
845
RDGSLTS
1050
RSDHLTT
1255
1000


 


600
GAGTCGGAA
641
QSANLAR
846
RSDELRT
1051
RSDNLAR
1256
206.7


 


601
GAGTCGGAA
642
RSANLTR
847
RLDGLRT
1052
RSDNLAR
1257
606.7


 


602
GAGTCGGAA
643
RSANLTR
848
RQDTLVG
1053
RSDNLAR
1258
616.7


 


603
GAGTCGGAA
644
QSGNLAR
849
RSDELRT
1054
RSDNLAR
1259
166.7


 


606
GGGGAGGATC
645
TTSNLRR
850
RSDNLQR
1055
RSDHLSR
1260
0.2


























TABLE 3







SEQ

SEQ

SEQ

SEQ
Kd



SBS#
TARGET
ID
F1
ID
F2
ID
F3
ID
(nM)

























897
GAGGAGGTGA
1261
RSDALAR
1347
RSDNLAR
1433
RSDNLVR
1519
0.07



 


828
GCGGAGGACC
1262
EKANLTR
1348
RSDNLAR
1434
RSDERKR
1520
0.1


 


884
GAGGAGGTGA
1263
RSDSLTR
1349
RSDNLAR
1435
RSDNLVR
1521
0.15


 


817
GAGGAGGTGA
1264
RSDSLTR
1350
RSDNLAR
1436
RSDNLAR
1522
0.31


 


666
GCGGAGGCGC
1265
RSDDLTR
1351
RSDNLTR
1437
RSDTLKK
1523
0.5


 


829
GCGGAGGACC
1266
EKANLTR
1352
RSDNLAR
1438
RSDTLKK
1524
0.52


 


670
GACGTGGAGG
1267
RSDNLAR
1353
RSDALAR
1439
DRSNLTR
1525
0.57


 


801
AAGGAGTCGC
1268
RSADLRT
1354
RSDNLAR
1440
RSDNLTQ
1526
0.85


 


668
GTGGAGGCCA
1269
ERGTLAR
1355
RSDNLAR
1441
RSDALAR
1527
1.13


 


895
ATGGATTCAG
1270
QSHDLTK
1356
TSGNLVR
1442
RSDALTQ
1528
1.4


 


799
GGGGGAGCTG
1271
QSSDLQR
1357
QRAHLER
1443
RSDHLSR
1529
1.85


 


798
GGGGGAGCTG
1272
QSSDLQR
1358
QSGHLQR
1444
RSDHLSR
1530
3


 


842
GAGGTGGGCT
1273
DRSHLTR
1359
RSDALAR
1445
RSDNLAR
1531
5.4


 


894
TCAGTGGTAT
1274
QRSALAR
1360
RSDALSR
1446
QSHDLTK
1532
6.15


 


892
ATGGATTCAG
1275
QSHDLTK
1361
QQSNLVR
1447
RSDALTQ
1533
6.2


 


888
TCAGTGGTAT
1276
QSSSLVR
1362
RSDALSR
1448
QSHDLTK
1534
14


 


739
GCGGGCGGGC
1277
RSDHLTR
1363
ERGHLTR
1449
RSDDLRR
1535
16.5


 


850
CAGGCTGTGG
1278
RSDALTR
1364
QSSDLTR
1450
RSDNLRE
1536
17


 


797
GCAGAGGCTG
1279
QSSDLQR
1365
RSDNLAR
1451
QSGDLTR
1537
17.5


 


891
TCAGTGGTAT
1280
QSSSLVR
1366
RSDALSR
1452
QSGSLRT
1538
18.5


 


887
TCAGTGGTAT
1281
QRSALAR
1367
RSDALSR
1453
QSGDLRT
1539
23.75


 


672
TCGGACGTGG
1282
RSDALAR
1368
DRSNLTR
1454
RSDELRT
1540
24


 


836
GGGGAGGCCC
1283
ERGTLAR
1369
RSDNLAR
1455
RSDHLSR
1541
24.25


 


674
GCGGCGTCGG
1284
RSDELRT
1370
RADTLRR
1456
RSDTLKK
1542
27.5


 


849
GGGGCCCTGG
1285
RSDALRE
1371
DRSSLTR
1457
RSDHLTQ
1543
29.05


 


825
GAATGGGCAG
1286
QSGSLTR
1372
RSDHLTT
1458
QSGNLTR
1544
37.3


 


673
GCGGGTGTCT
1287
DRSALAR
1373
QSSHLAR
1459
RSDTLKK
1545
48.33


 


848
GGGGAGGCCC
1288
DRSSLTR
1374
RSDNLAR
1460
RSDHLSR
1546
49.5


 


662
AGAGCGGCAC
1289
QTGSLTR
1375
RSDELQR
1461
QSGHLNQ
1547
50


 


667
GAGTCGGACG
1290
DRSNLTR
1376
RSDELRT
1462
RSDNLAR
1548
50


 


803
GCAGCGGCTC
1291
QSSDLQR
1377
RSDELQR
1463
QSGSLTR
1549
57.5


 


671
TCGGACGAGT
1292
RSDNLAR
1378
DRSNLTR
1464
RSDELRT
1550
64


 


851
GAGATGGATC
1293
QSSNLQR
1379
RRDVLMN
1465
RLHNLQR
1551
74


 


804
GCAGCGGCTC
1294
QSSDLQR
1380
RSDDLNR
1466
QSGSLTR
1552
82.5


 


669
GACGAGTCGG
1295
RSDELRT
1381
RSDNLAR
1467
DRSNLTR
1553
90


 


682
GCTGCAGGAG
1296
RSDHLAR
1382
QSGDLTR
1468
QSSDLSR
1554
90


 


845
GAGATGGATC
1297
QSSNLQR
1383
RSDALRQ
1469
RLHNLQR
1555
112.5


 


663
AGAGCGGCAC
1298
QTGSLTR
1384
RSDELQR
1470
KNWKLQA
1556
115


 


738
GCGGGGTCCG
1299
ERGTLTT
1385
RSDHLSR
1471
RSDDLRR
1557
120


 


664
AGAGCGGCAC
1300
QTGSLTR
1386
RADTLRR
1472
ASSRLAT
1558
125


 


833
GACTAGGACC
1301
EKANLTR
1387
RSDNLTK
1473
DRSNLTR
1559
136


 


685
GCTGCAGGAG
1302
RSDHLAR
1388
QSGSLTR
1474
QSSDLSR
1560
150


 


835
TAGGGAGCGT
1303
RADTLRR
1389
QSGHLTR
1475
RSDNLTT
1561
150


 


847
TAGGGAGCGT
1304
RSDDLTR
1390
QSGHLTR
1476
RSDNLTT
1562
150


 


818
GAATGGGCAG
1305
QSGSLTR
1391
RSDHLTT
1477
QSSNLVR
1563
167


 


834
GACTAGGACC
1306
EKANLTR
1392
RSDHLTT
1478
DRSNLTR
1564
186


 


837
GGGGCCCTGG
1307
RSDALRE
1393
DRSSLTR
1479
RSDHLSR
1565
222


 


764
GCAGAGGCTG
1308
TSGELVR
1394
RSDNLAR
1480
QSGDLTR
1566
255


 


774
GCAGCGGTAG
1309
QRSALAR
1395
RSDELQR
1481
QSGDLTR
1567
258


 


765
GCCGAGGCCG
1310
ERGTLAR
1396
RSDNLAR
1482
ERGTLAR
1568
262.5


 


766
GCCGAGGCCG
1311
ERGTLAR
1397
RSDNLAR
1483
DRSDLTR
1569
262.5


 


775
GCAGCGGTAG
1312
QSGALTR
1398
RSDELQR
1484
QSGDLTR
1570
265


 


763
GCAGAGGCTG
1313
TSGELVR
1399
RSDNLAR
1485
QSGSLTR
1571
275


 


838
GGGGCCCTGG
1314
RSDALRE
1400
DRSSLTR
1486
RSDHLTA
1572
300


 


841
GAGTGTGAGG
1315
RSDNLAR
1401
QSSHLAS
1487
RSDNLAR
1573
300


 


770
TTCGCAGCCT
1316
DRSSLTR
1402
QSGSLTR
1488
RSDSLTK
1574
325


 


767
GGGGGAGCTG
1317
QSSDLAR
1403
QSGHLQR
1489
RSDHLSR
1575
335


 


800
TTGGCAGCCT
1318
ERGTLAR
1404
QSGSLTR
1490
RSDSLTK
1576
400


 


832
GACTAGGACC
1319
EKANLTR
1405
RSDNLTT
1491
DRSNLTR
1577
408


 


844
GAGATGGATC
1320
QSSNLQR
1406
RSDALRQ
1492
RSDNLQR
1578
444


 


683
GCTGCAGGAG
1321
QSGHLAR
1407
QSGSLTR
1493
QSSDLSR
1579
500


 


805
GCAGCGGTAG
1322
QRSALAR
1408
RSDELQR
1494
QSGSLTR
1580
500


 


839
GAGTGTGAGG
1323
RSDNLAR
1409
TSDHLAS
1495
RSDNLAR
1581
625


 


840
GAGTGTGAGG
1324
RSDNLAR
1410
MSHHLKT
1496
RSDNLAR
1582
625


 


830
GGAGAGTCGG
1325
RSDELRT
1411
RSDNLAR
1497
QPAHLAR
1583
683


 


831
GGAGAGTCGG
1326
RSDDLTK
1412
RSDNLAR
1498
QRAHLAR
1584
700


 


684
GCTGCAGGAG
1327
RSAHLAR
1413
QSGSLTR
1499
QSSDLSR
1585
850


 


846
GAGATGGATC
1328
QSSNLQR
1414
RRDVLNN
1500
RSDNLQR
1586
889.5


 


819
AAGTAGGGTG
1329
QSSHLTR
1415
RSDNLTT
1501
RSDNLTQ
1587
1000


 


820
ACGGTAGTTA
1330
QSSALTR
1416
QRSALAR
1502
RSDTLTQ
1588
1000


 


821
ACGGTAGTTA
1331
NRATLAR
1417
QRSALAR
1503
RSDTLTQ
1589
1000


 


822
GTGTGCTGGT
1332
RSDHLTT
1418
ERQHLAT
1504
RSDALAR
1590
1000


 


823
GTGTGCTGGT
1333
RSDHLTK
1419
ERQHLAT
1505
RSDALAR
1591
1000


 


824
GTGTGCTGGT
1334
RSDHLTT
1420
DRSHLRT
1506
RSDALAR
1592
1000


 


885
GTGTGCTGGT
1335
RSDHLTK
1421
DRSHLRT
1507
RSDALAR
1593
1000


 


886
TCAGTGGTAT
1336
QSSSLVR
1422
RSDALSR
1508
QSGDLRT
1594
1000


 


889
ATGGATTCAG
1337
QSGSLTT
1423
QQSNLVR
1509
RSDALTQ
1595
1000


 


890
CTGGTATGTC
1338
QRSHLTT
1424
QRSALAR
1510
RSDALRE
1596
1000


 


896
AAGTAGGGTG
1339
TSGHLVR
1425
RSDNLTT
1511
RSDNLTQ
1597
1000


 


898
ACGGTAGTTA
1340
NPATLAR
1426
QSSSLVR
1512
RSDTLTQ
1598
1000


 


899
CTGGTATGTC
1341
QRSHLTT
1427
QSSSLVR
1513
RSDALRE
1599
1000


 


900
CTGGTATGTC
1342
MSHHLKE
1428
QSSSLVR
1514
RSDALRE
1600
1000


 


901
CTGGTATGTC
1343
NSHHLKE
1429
QRSALAR
1515
RSDALRE
1601
1000


 


773
GCAGCGGTAG
1344
QSGALTR
1430
RSDELQR
1516
QSGSLTR
1602
1250


 


768
GGGGGAGCTG
1345
QSSDLAR
1431
QRAHLER
1517
RSDHLSR
1603
2000


 


681
GCTGCAGGAG
1346
RSAHLAR
1432
QSGDLTR
1518
QSSDLSR
1604
3000


























TABLE 4







SEQ

SEQ

SEQ

SEQ
Kd



SBS#
TARGET
ID
F1
ID
F2
ID
F3
ID
(nM)

























607
AAGGTGGCAG
1605
QSGDLTR
1707
RSDSLAR
1809
RLDNRTA
1911
6.5



 


608
TTGGCTGGGC
1606
GSWHLTR
1708
QSSDLQR
1810
RSDSLTK
1912
8


 


611
GTGGCTGCAG
1607
QSGDLTR
1709
QSSDLQR
1811
RSDALAR
1913
11.5


 


612
GTGGCTGCAG
1608
QSGTLTR
1710
QSSDLQR
1812
RSDALAR
1914
0.38


 


613
TTGGCTGGGC
1609
RSDHLAR
1711
QSSDLQR
1813
RGDALTS
1915
1.45


 


614
TTGGCTGGGC
1610
RSDHLAR
1712
QSSDLQR
1814
RSDSLTK
1916
2


 


616
GAGGAGGATG
1611
QSSNLQR
1713
RSDNLAR
1815
RSDNLQR
1917
0.08


 


617
AAGGGGGGG
1612
RSDHLSR
1714
RSDHLTR
1816
RKDNMTA
1918
1


 


618
AAGGGGGGG
1613
RSDHLSR
1715
RSDHLTR
1817
RKDNNTQ
1919
0.55


 


619
AAGGGGGGG
1614
RSDHLSR
1716
RSDHLTR
1818
RKDNMTN
1920
1.34


 


620
AAGGGGGGG
1615
RSDHLSR
1717
RSDHLTR
1819
RLDNRTA
1921
0.54


 


621
AAGGGGGGG
1616
RSDHLSR
1718
RSDHLTR
1820
RLDNRTQ
1922
0.75


 


624
ACGGATGTCT
1617
DRSALAR
1719
TSANLAR
1821
RSDTLRS
1923
7


 


628
TTGTAGGGGA
1618
RSDHLTR
1720
RSDNLTT
1822
RGDALTS
1924
130


 


629
TTGTAGGGGA
1619
RSSHLTR
1721
RSDNLTT
1823
RGDALTS
1925
150


 


630
CGGGGAGAGT
1620
RSDNLAR
1722
QSGHLQR
1824
RSDHLRE
1926
37.5


 


646
TTGGTGGAAG
1621
QSGNLAR
1723
RSDALAR
1825
RGDALTS
1927
35


 


647
TTGGTGGAAG
1622
QSANLAR
1724
RSDALAR
1826
RGDALTS
1928
40


 


651
GTTGTGGAAT
1623
QSGNLSR
1725
RSDALAR
1827
NRATLAR
1929
67.5


 


652
TAGGAGGCTG
1624
QSSDLQR
1726
RSDNLAR
1828
RSDNLTT
1930
1.5


 


653
TAGGAGGCTG
1625
TTSDLTR
1727
RSDNLAR
1829
RSDNLTT
1931
5.5


 


654
TAGGCATAAA
1626
QSGNLRT
1728
QSGSLTR
1830
RSDNLTT
1932
105


 


655
TAGGCATAAA
1627
QSGNLRT
1729
QSSTLRR
1831
RSDNLTT
1933
1000


 


656
TAGGCATAAA
1628
QSGNLRT
1730
QSGSLTR
1832
RSDNLTS
1934
540


 


657
TAGGCATAAA
1629
QSGNLRT
1731
QSSTLRR
1833
RSDNLTS
1935
300


 


660
GAGGGAGTTC
1630
NRATLAR
1732
QSGHLTR
1834
RSDNLAR
1936
8.25


 


661
GAGGGAGTTC
1631
TTSALTR
1733
QSGHLTR
1835
RSDNLAR
1937
1.73


 


665
GCGGAGGCGC
1632
RSDDVTR
1734
RSDNLTR
1836
RSDDLRR
1938
12.5


 


689
AAGGCGGAGA
1633
RSDNLTR
1735
RSDELQR
1837
RLDNRTA
1939
82.5


 


692
AAGGCGGAGA
1634
RSDNLTR
1736
RSDELQR
1838
RSDNLTQ
1940
51


 


693
AAGGCGGAGA
1635
RSDNLTR
1737
RADTLRR
1839
RLDNRTA
1941
95


 


694
AAGGCGGAGA
1636
RSDNLTR
1738
EADTLRR
1840
RSDNLTQ
1942
28.5


 


695
GGGGGCGAGC
1637
RSSNLTR
1739
DRSHLAR
1841
RSDHLTR
1943
850


 


697
TGAGCGGCGG
1638
RSDELTR
1740
RSDELSR
1842
QSGHLTK
1944
200


 


698
TGAGCGGCGG
1639
RSDELTR
1741
RSDELSR
1843
QSHGLTS
1945
300


 


699
GCGGCGGCAG
1640
QSGSLTR
1742
RSDDLQR
1844
RSDERKR
1946
21.5


 


700
GCGGCGGCAG
1641
QSGDLTR
1743
RSDDLQR
1845
RSDERKR
1947
45


 


701
GCAGCGGAGC
1642
RSDNLAR
1744
RSDELQR
1846
QSGSLTR
1948
50.5


 


702
GCAGCGGAGC
1643
RSDNLAR
1745
RSDELQR
1847
QSGDLTR
1949
73.5


 


704
AAGGTGGCAG
1644
QSGDLTR
1746
RSDSLAR
1848
RSDNLTQ
1950
5


 


705
GGGGTGGGGC
1645
RSDHLAR
1747
RSDSLAR
1849
RSDHLSR
1951
0.01


 


706
GGGGTGGGGC
1646
RSDHLAR
1748
RSDSLLR
1850
RSDHLSR
1952
0.05


 


708
GAGTCGGAA
1647
QSANLAR
1749
RQDTLVG
1851
RSDNLAR
1953
300


 


709
GAGTCGGAA
1648
QSANLAR
1750
RKDVLVS
1852
RSDNLAR
1954
400


 


710
GAGTCGGAA
1649
QSGNLAR
1751
RLDGLRT
1853
RSDNLAR
1955
400


 


711
GAGTCGGAA
1650
QSGNLAR
1752
RQDTLVG
1854
RSDNLAR
1956
400


 


712
GGTGAGGAGT
1651
RSDNLAR
1753
RSDNLAR
1855
MSDHLSR
1957
9.5


 


713
GGTGAGGAGT
1652
RSDNLAR
1754
RSDNLAR
1856
MSHHLSR
1958
0.15


 


714
TGGGTCGCGG
1653
RSDELRR
1755
DRSALAR
1857
RSDHLTT
1959
200


 


715
TGGGTCGCGG
1654
RADTLRR
1756
DRSALAR
1858
RSDHLTT
1960
0.46


 


716
TTGGGAGCAC
1655
QSGSLTR
1757
QSGHLQR
1859
RGDALTS
1961
200


 


717
TTGGGAGCAC
1656
QSGSLTR
1758
QSGHLQR
1860
RSDALTK
1962
150


 


718
TTGGGAGCAC
1657
QSGSLTR
1759
QSGHLQR
1861
RSDALTR
1963
107.5


 


719
GGCATGGTGG
1658
RSDALTR
1760
RSDALTS
1862
DRSHLAR
1964
20


 


720
GAAGAGGATG
1659
TTSNLAR
1761
RSDNLAR
1863
QSGNLTR
1965
1.6


 


722
ATGGGGGTGG
1660
RSDALTR
1762
RSDHLTR
1864
RSDALRQ
1966
0.7


 


724
GGCATGGTGG
1661
RSDALTR
1763
RSDALRQ
1865
DRSHLAR
1967
2.5


 


725
GCTTGAGTTA
1662
QSSALAR
1764
QSGHLQK
1866
QSSDLQR
1968
3000


 


726
GAAGAGGATG
1663
QSSNLAR
1765
RSDNLAR
1867
QSGNLTR
1969
1.5


 


727
GCGGTGGCTC
1664
QSSDLTR
1766
RSDALSR
1868
RSDTLKK
1970
0.1


 


728
GGTGAGGAGT
1665
RSDNLAR
1767
RSDNLAR
1869
DSSKLSR
1971
15


 


729
GGAGGGGAGT
1666
RSDNLAR
1768
RSDHLSR
1870
QSGHLAR
1972
1000


 


730
TGGGTCGCGG
1667
RSDDLTR
1769
DRSALAR
1871
RSDHLTT
1973
1000


 


731
GTGGGGGAGA
1668
RSDNLAR
1770
RSDHLSR
1872
RSDALAR
1974
12


 


732
GCGGGTGGGG
1669
RSDHLAR
1771
QSSHLAR
1873
RSDDLTR
1975
22.5


 


733
GCGGGTGGGG
1670
RSDHLAR
1772
QSSHLAR
1874
RSDTLKK
1976
0.32


 


734
GGGGCTGGGT
1671
RSDHLAR
1773
QSSDLSR
1875
RSDHLSR
1977
0.25


 


735
GCGGTGGCTC
1672
QSSDLTR
1774
RSDALSR
1876
RSDERKR
1978
0.05


 


736
GAGGTGGGGA
1673
RSDHLAR
1775
RSDALSR
1877
RSDNLSR
1979
0.47


 


737
GGAGGGGAGT
1674
RSDNLAR
1776
RSDHLSR
1878
QRGHLSR
1980
1000


 


740
AAGGTGGCAG
1675
QSGSLTR
1777
RSDALAR
1879
RSDNRTA
1981
12.5


 


741
AAGGCTGAGA
1676
RSDNLTR
1778
QSSDLQR
1880
RSDNLTQ
1982
15


 


742
ACGGGGTTAT
1677
QRGALAS
1779
RSDHLSR
1881
RSDTLKQ
1983
29


 


743
ACGGGGTTAT
1678
QRGALAS
1780
RSDHLSR
1882
RSDTLTQ
1984
10


 


744
ACGGGGTTAT
1679
QRSALAS
1781
RSDHLSR
1883
RSDTLKQ
1985
8.33


 


745
ACGGGGTTAT
1680
QRSALAS
1782
RSDHLSR
1884
RSDTLTQ
1986
12.5


 


746
CTGGAAGCAT
1681
QSGSLTR
1783
QSGNLAR
1885
RSDALRE
1987
2.07


 


747
CTATTTTGGG
1682
RSDHLTT
1784
QSSALRT
1886
QSGALRE
1988
2000


 


748
TTGGACGGCG
1683
DSGHLTR
1785
DRSNLER
1887
RGDALTS
1989
112.3


 


749
TTGGACGGCG
1684
DRSHLTR
1786
DSSNLTR
1888
RGDALTS
1990
11.33


 


750
GAGGGAGCGA
1685
RSDELTR
1787
QSAHLAR
1889
RSDNLAR
1991
52


 


751
GGTGAGGAGT
1686
RSDNLAR
1788
RSDNLAR
1890
NRSHLAR
1992
7


 


752
GAGGTGGGGA
1687
RSHHLAR
1789
RSDALSR
1891
RSDNLSR
1993
31


 


757
CGGGCGGCTG
1688
QSSDLRR
1790
RSDELQR
1892
RSDHLRE
1994
14.5


 


758
CGGGCGGCTG
1689
QSSDLRR
1791
RADTLRR
1893
RSDHLRE
1995
16.5


 


759
TTGGACGGCG
1690
DSGHLTR
1792
DSSNLTR
1894
RGDALTS
1996
37


 


760
TTGGACGGCG
1691
DRSHLTR
1793
DRSNLER
1895
RGDALTS
1997
148.5


 


761
GCGGTGGCTC
1692
QSSDLQR
1794
RSDALSR
1896
RSDERKR
1998
6


 


762
GCGGTGGCTC
1693
QSSDLQR
1795
RSDALSR
1897
RSDTLKK
1999
18


 


776
ATGGACGGGT
1694
RSDHLAR
1796
DRSNLER
1898
RSDSLNQ
2000
0.4


 


777
ATGGACGGGT
1695
RSDHLAR
1797
DRSNLTR
1899
RSDALSA
2001
3.4


 


779
CGGGGAGCAG
1696
QSGSLTR
1798
QSGHLTR
1900
RSDHLAE
2002
0.5


 


780
CGGGGAGCAG
1697
QSGSLTR
1799
QSGHLTR
1901
RSDHLRA
2003
0.5


 


781
GGGGAGCAGC
1698
RSSNLRE
1800
RSDNLAR
1902
RSDHLTR
2004
4.25


 


783
TTGGGAGCGG
1699
RSDELTR
1801
QSGHLQR
1903
RGDALTS
2005
2000


 


785
TTGGGAGCGG
1700
RSDTLKK
1802
QSGHLQR
1904
RSDALTS
2006
50


 


786
TTGGGAGCGG
1701
RSDTLKK
1803
QSGHLQR
1905
RGDALRS
2007
2000


 


787
AGGGAGGATG
1702
QSDNLAR
1804
RSDNLAR
1906
RSDHLTQ
2008
4


 


826
GAGGGAGCGA
1703
RSDELTR
1805
QSGHLAR
1907
RSDNLAR
2009
2.75


 


827
GAGGGAGCGA
1704
RADTLRR
1806
QSGHLAR
1908
RSDNLAR
2010
1.2


 


882
GCGTGGGCGT
1705
RSDELTR
1807
RSDHLTT
1909
RSDERKR
2011
0.01


 


883
CCGTGGGCGT
1706
RSDELTR
1808
RSDHLTT
1910
RSDERKR
2012
1


























TABLE 5







SEQ

SEQ

SEQ

SEQ
Kd



SBS#
TARGET
ID
F1
ID
F2
ID
F3
ID
(Nm)

























903
ATGGAAGGG
2013
RSDHLAR
2513
QSGNLAR
3013
RSDALRQ
3513
1.027



 


904
AAGGGTGAC
2014
DSSNLTR
2514
QSSHLAR
3014
RSDNLTQ
3514
1


 


905
GTGGTGGTG
2015
RSSALTR
2515
RSDSLAR
3015
RSDSLAR
3515
1.15


 


908
AAGGTCTCA
2016
QSGDLRT
2516
DRSALAR
3016
RSDNLRQ
3516
50


 


909
GTGGAAGAA
2017
QSGNLSR
2517
QSGNLQR
3017
RSDALAR
3517
16.4


 


910
ATGGAAGAT
2018
QSSNLAR
2518
QSGNLQR
3018
RSDALAQ
3518
0.03


 


911
ATGGGTGCA
2019
QSGSLTR
2519
QSSHLAR
3019
RSDALAQ
3519
0.91


 


912
TCAGAGGTG
2020
RSDSLAR
2520
RSDNLTR
3020
QSGDLRT
3520
0.135


 


914
CAGGAAAAG
2021
RSDNLTQ
2521
QSGNLAR
3021
RSDNLRE
3521
1.26


 


915
CAGGAAAAG
2022
RSDNLRQ
2522
QSGNLAR
3022
RSDNLRE
3522
45.15


 


916
GAGGAAGGA
2023
QSGHLAR
2523
QSGNLAR
3023
RSDNLQR
3523
1.3


 


919
TCATAGTAG
2024
RSDNLTT
2524
RSDNLRT
3024
QSGDLRT
3524
250


 


920
GATGTGGTA
2025
QSSSLVR
2525
RSDSLAR
3025
TSANLSR
3525
4


 


921
AAGGTCTCA
2026
QSGDLRT
2526
DPGALVR
3026
RSDNLRQ
3526
11


 


922
AAGGTCTCA
2027
QSHDLTK
2527
DRSALAR
3027
RSDNLRQ
3527
4


 


923
AAGGTCTCA
2028
QSHDLTK
2528
DPGALVR
3028
RSDNLRQ
3528
2


 


926
GTGGTGGTG
2029
RSDALTR
2529
RSDSLAR
3029
RSDSLAR
3529
7.502


 


927
CAGGTTGAG
2030
RSDNLAR
2530
TSGSLTR
3030
RSDNLRE
3530
3.61


 


928
CAGGTTGAG
2031
RSDNLAR
2531
QSSALTR
3031
RSDNLRE
3531
25


 


929
CAGGTAGAT
2032
QSSNLAR
2532
QSATLAR
3032
RSDNLRE
3532
1.3


 


931
GAGGAAGAG
2033
RSDNLAR
2533
QSSNLVR
3033
RSDNLAR
3533
2


 


932
ATGGAAGGG
2034
RSDHLAR
2534
QSSNLVR
3034
RSDALRQ
3534
797


 


933
GACGAGGAA
2035
QSANLAR
2535
RSDNLAR
3035
DRSNLTR
3535
500


 


934
ATGGAAGAT
2036
QSSNLAR
2536
QSGNLQR
3036
RSDALTS
3536
0.07


 


935
ATGGGTGCA
2037
QSGSLTR
2537
QSSHLAR
3037
RSDALTS
3537
0.91


 


937
GTGGGGGCT
2038
QSSDLTR
2538
RSDHLTR
3038
RSDSLAR
3538
0.03


 


938
GTGGGGGCT
2039
QSSDLRR
2539
RSDHLTR
3039
RSDSLAR
3539
0.049


 


939
GGGGGCTGG
2040
RSDHLTT
2540
DRSHLAR
3040
RSDHLSK
3540
0.352


 


940
GGGGGCTGG
2041
RSDHLTK
2541
DRSHLAR
3041
RSDHLSK
3541
1.5


 


941
GGGGCTGGG
2042
RSDHLAR
2542
QSSDLRR
3042
RSDKLSR
3542
0.077


 


942
GGGGCTGGG
2043
RSDHLAR
2543
QSSDLRR
3043
RSDHLSK
3543
0.13


 


943
GGGGCTGGG
2044
RSDHLAR
2544
TSGELVR
3044
RSDKLSR
3544
0.067


 


944
GGGGCTGGG
2045
RSDHLAR
2545
TSGELVR
3045
RSDHLSK
3545
0.027


 


945
GGTGCGGTG
2046
RSDSLTR
2546
RADTLRR
3046
MSHHLSR
3546
0.027


 


946
GGTGCGGTG
2047
RSDSLTR
2547
RSDVLQR
3047
MSHHLSR
3547
0.027


 


947
GGTGCGGTG
2048
RSDSLTR
2548
RSDELQR
3048
QSSHLAR
3548
0.013


 


948
GGTGCGGTG
2049
RSDSLTR
2549
RSDVLQR
3049
QSSHLAR
3549
0.017


 


962
GAGGCGGCA
2050
QSGSLTR
2550
RSDELQR
3050
RSDNLAR
3550
0.015


 


963
GAGGCGGCA
2051
QSGSLTR
2551
RSDDLQR
3051
RSDNLAR
3551
0.015


 


964
GCGGCGGTG
2052
RSDALAR
2552
RSDELQR
3052
RSDERKR
3552
0.041


 


965
GCGGCGGCC
2053
ERGDLTR
2553
RSDELQR
3053
RSDERKR
3553
3.1


 


966
GAGGAGGCC
2054
ERGTLAR
2554
RSDNLSR
3054
RSDNLAR
3554
0.028


 


967
GAGGAGGCC
2055
DRSSLTR
2555
RSDNLSR
3055
RSDNLAR
3555
0.055


 


968
GAGGCCGCA
2056
QSGSLTR
2556
DRSSLTR
3056
RSDNLAR
3556
1.4


 


969
GAGGCCGCA
2057
QSGSLTR
2557
DRSDLTR
3057
RSDNLAR
3557
0.275


 


970
GTGGGCGCC
2058
ERGTLAR
2558
DRSHLAR
3058
RSDALAR
3558
1.859


 


971
GTGGGCGCC
2059
DRSSLTR
2559
DRSHLAR
3059
RSDALAR
3559
0.144


 


972
GTGGGCGCC
2060
ERGDLTR
2560
DRSHLAR
3060
RSDALAR
3560
1.748


 


973
GCCGCGGTC
2061
DRSALTR
2561
RSDELQR
3061
ERGTLAR
3561
0.6


 


974
GCCGCGGTC
2062
DRSALTR
2562
RSDELQR
3062
DRSDLTR
3562
0.038


 


975
CAGGCCGCT
2063
QSSDLTR
2563
DRSSLTR
3063
RSDNLRE
3563
1.1


 


976
CAGGCCGCT
2064
QSSDLTR
2564
DRSDLTR
3064
RSDNLRE
3564
4.12


 


977
CTGGCAGTG
2065
RSDSLTR
2565
QSGSLTR
3065
RSDALRE
3565
0.017


 


978
CTGGCAGTG
2066
RSDSLTR
2566
QSGDLTR
3066
RSDALRE
3566
1.576


 


979
CTGGCGGCG
2067
RSSDLTR
2567
RSDELQR
3067
RSDALRE
3567
1.59


 


980
CTGGCGGCG
2068
RSDDLTR
2568
RSDELQR
3068
RSDALRE
3568
2.2


 


981
CAGGCGGCG
2069
RSDDLTR
2569
RSDELQR
3069
RSDNLRE
3569
0.375


 


982
CCGGGCTGG
2070
RSDHLTT
2570
DRSHLAR
3070
RSDELRE
3570
0.03


 


983
CCGGGCTGG
2071
RSDHLTK
2571
DRSHLAR
3071
RSDELRE
3571
1.385


 


984
GACGGCGAG
2072
RSDNLAR
2572
DRSHLAR
3072
DRSNLTR
3572
1.6


 


985
GACGGCGAG
2073
RSDNLAR
2573
DRSHLAR
3073
EKANLTR
3573
0.965


 


986
GGTGCTGAT
2074
QSSNLQR
2574
QSSDLQR
3074
MSHHLSR
3574
1.6


 


987
GGTGCTGAT
2075
QSSNLQR
2575
QSSDLQR
3075
TSGHLVR
3575
33.55


 


988
GGTGCTGAT
2076
TSGNLVR
2576
QSSDLQR
3076
MSHHLSR
3576
0.15


 


989
GGTGAGGGG
2077
RSDHLAR
2577
RSDNLAR
3077
MSHHLSR
3577
1.9


 


990
AAGGTGGGC
2078
DRSHLTR
2578
RSDSLAR
3078
RSDNLTQ
3578
5.35


 


991
AAGGTGGGC
2079
DRSHLTR
2579
SSGSLVR
3079
RSDNLTQ
3579
0.06


 


993
GGGGCTGGG
2080
RSDHLAR
2580
TSGELVR
3080
RSDHLSR
3580
3.1


 


994
GGGGGCTGG
2081
RSDHLTK
2581
DRSHLAR
3081
RSDHLSR
3581
0.03


 


995
GGGGAGGAA
2082
QSANLAR
2582
RSDNLAR
3082
RSDHLSK
3582
0.08


 


996
CAGTTGGTC
2083
DRSALAR
2583
RSDALTS
3083
RSDNLRE
3583
9.6


 


997
AGAGAGGCT
2084
QSSDLTR
2584
RSDNLAR
3084
QSGHLNQ
3584
1.65


 


998
ACGTAGTAG
2085
RSANLRT
2585
RSDNLTK
3085
RSDTLKQ
3585
0.23


 


999
AGAGAGGCT
2086
QSSDLTR
2586
RSDNLAR
3086
QSGKLTQ
3586
0.6


 


1000
CAGTTGGTC
2087
DRSALAR
2587
RSDALTR
3087
RSDNLRE
3587
11.15


 


1001
GGAGCTGAC
2088
EKANLTR
2588
QSSDLSR
3088
QPAHLAR
3588
1.8


 


1002
GCGGAGGAG
2089
RSDNLVR
2589
RSDNLAR
3089
RSDERKR
3589
0.028


 


1003
ACGTAGTAG
2090
RSANLRT
2590
RSDNLTK
3090
RSDTLRS
3590
0.118


 


1004
ACGTAGTAG
2091
RSDNLTT
2591
RSDNLTK
3091
RSDTLRS
3591
1.4


 


1006
GTAGGGGCG
2092
RSDDLTR
2592
RSDHLTR
3092
QRASLTR
3592
0.898


 


1007
GAGAGAGAT
2093
QSSNLQR
2593
QSGHLTR
3093
RLHNLAR
3593
167


 


1008
GAGATGGAG
2094
RSDNLSR
2594
RSDSLTQ
3094
RLHNLAR
3594
0.4


 


1009
GAGATGGAG
2095
RSDNLSR
2595
RSDSLTQ
3095
RSDNLSR
3595
1.9


 


1010
GAGAGAGAT
2096
QSSNLQR
2596
QSGHLTR
3096
RSDNLAR
3596
8.2


 


1011
TTGGTGGCG
2097
RSADLTR
2597
RSDSLAR
3097
RSDSLTK
3597
0.03


 


1012
GACGTAGGG
2098
RSDHLTR
2598
QSSSLVR
3098
DRSNLTR
3598
0.032


 


1013
GAGAGAGAT
2099
QSSNLQR
2599
QSGHLNQ
3099
RSDNLAR
3599
0.15


 


1014
GACGTAGGG
2100
RSDHLTR
2600
QSGSLTR
3100
DRSNLTR
3600
0.01


 


1015
GCGGAGGAG
2101
RSDNLVR
2601
RSDNLAR
3101
RSDTLKK
3601
0.008


 


1016
CAGTTGGTC
2102
DRSALAR
2602
RSDSLTK
3102
RSDNLRE
3602
0.09


 


1017
CTGGATGAC
2103
EKANLTR
2603
TSGNLVR
3103
RSDALRE
3603
0.233


 


1018
GTAGTAGAA
2104
QSANLAR
2604
QSSSLVR
3104
QPASLAR
3604
7.2


 


1019
AGGGAGGAG
2105
RSDNLAR
2605
RSDNLAR
3105
RSDHLTQ
3605
0.022


 


1020
ACGTAGTAG
2106
RSDNLTT
2606
RSDNLTK
3106
RSDTLKQ
3606
0.69


 


1022
GAGGAGGTG
2107
RSDALAR
2607
RSDNLAR
3107
RSDNLAR
3607
0.01


 


1024
GGGGAGGAA
2108
QSANLAR
2608
RSDNLAR
3108
RSDHLSR
3608
0.08


 


1025
GAGGAGGTG
2109
QSSALTR
2609
QSSSLVR
3109
RSDTLTQ
3609
0.115


 


1026
GTGGCTTGT
2110
MSHHLKE
2610
QSSDLSR
3110
RSDALAR
3610
0.076


 


1027
GCGGCGGTG
2111
RSDALAR
2611
RSDELQR
3111
RSDELQR
3611
0.054


 


1032
GGTGCTGAT
2112
TSGNLVR
2612
QSSDLQR
3112
TSGHLVR
3612
0.52


 


1033
GTGTTCGTG
2113
RSDALAR
2613
DRSALTT
3113
RSDALAR
3613
685.2


 


1034
GTGTTCGTG
2114
RSDALAR
2614
DRSALTK
3114
RSDALAR
3614
14.55


 


1035
GTGTTCGTG
2115
RSDALAR
2615
DRSALRT
3115
RSDALAR
3615
56


 


1037
GTAGGGGCA
2116
QSGSLTR
2616
RSDHLSR
3116
QRASLAR
3616
0.05


 


1038
GTAGGGGCA
2117
QTGELRR
2617
RSDHLSR
3117
QPASLAR
3617
0.152


 


1039
GGGGCTGGG
2118
RSDHLSR
2618
TSGELVR
3118
RSDHLTR
3618
1.37


 


1040
GGGGCTGGG
2119
RSDHLSR
2619
QSSDLQR
3119
RSDHLSK
3619
0.05


 


1041
TCATAGTAG
2120
RSDNLTT
2620
RSDNLRT
3120
QSHDLTK
3620
2.06


 


1043
CAGGGAGAG
2121
RSDNLAR
2621
QSGHLTR
3121
RSDNLRE
3621
0.16


 


1044
CAGGGAGAG
2122
RSDNLAR
2622
QRAHLER
3122
RSDNLRE
3622
1.07


 


1045
GGGGCAGGA
2123
QSGHLAR
2623
QSGSLTR
3123
RSDHLSR
3623
0.15


 


1046
GGGGCAGGA
2124
QSGHLAR
2624
QSGDLRR
3124
RSDHLSR
3624
0.09


 


1047
GGGGCAGGA
2125
QRAHLER
2625
QSGSLTR
3125
RSDHLSR
3625
24.7


 


1048
CAGGCTGTA
2126
QSGALTR
2626
QSSDLQR
3126
RSDNLRE
3626
1.387


 


1049
CAGGCTGTA
2127
QRASLAR
2627
QSSDLQR
3127
RSDNLRE
3627
55.6


 


1050
CAGGCTGTA
2128
QSSSLVR
2628
QSSDLQR
3128
RSDNLRE
3628
0.125


 


1051
GAGGCTGAG
2129
RSDNLTR
2629
QSSDLQR
3129
RSDNLVR
3629
0.02


 


1052
TAGGACGGG
2130
RSDHLAR
2630
EKANLTR
3130
RSDNLTT
3630
0.28


 


1053
TAGGACGGG
2131
RSDHLAR
2631
DRSNLTR
3131
RSDNLTT
3631
0.025


 


1054
GCTGCAGGG
2132
RSDHLAR
2632
QSGSLTR
3132
QSSDLQR
3632
0.033


 


1055
GCTGCAGGG
2133
RSDHLAR
2633
QSGSLTR
3133
TSGDLTR
3633
18.73


 


1056
GCTGCAGGG
2134
RSDHLAR
2634
QSGSLTR
3134
QSSDLQR
3634
0.045


 


1057
GCTGCAGGG
2135
RSDHLAR
2635
QSGDLTR
3135
TSGDLTR
3635
0.483


 


1058
GGGGCCGCG
2136
RSDELTR
2636
DRSSLTR
3136
RSDHLSR
3636
6.277


 


1059
GGGGCCGCG
2137
RSDELTR
2637
DRSDLTR
3137
RSDHLSR
3637
0.152


 


1060
GCGGAGGCC
2138
ERGTLAR
2638
RSDNLAR
3138
RSDERKR
3638
0.69


 


1061
GTTGCGGGG
2139
RSDHLAR
2639
RSDELQR
3139
QSSALTR
3639
0.165


 


1062
GTTGCGGGG
2140
RSDHLAR
2640
RSDELQR
3140
TSGSLTR
3640
0.068


 


1063
GTTGCGGGG
2141
RSDHLAR
2641
RSDELQR
3141
MSHALSR
3641
0.96


 


1064
GCGGCAGTG
2142
RSDALTR
2642
QSGSLTR
3142
RSDERKR
3642
0.453


 


1065
TGGGGCGGG
2143
RSDHLAR
2643
DRSHLAR
3143
RSDHLTT
3643
1.37


 


1066
GAGGGCGGT
2144
QSSHLTR
2644
DRSHLAR
3144
RSDNLVR
3644
0.15


 


1067
GAGGGCGGT
2145
TSGHLVR
2645
DRSHLAR
3145
RSDNLVR
3645
1.37


 


1068
GCAGGGGGC
2146
DRSHLTR
2646
RSDHLTR
3146
QSGDLTR
3646
2.05


 


1069
GCAGGCGGT
2147
DRSHLTR
2647
RSDHLTR
3147
QSGSLTR
3647
0.1


 


1070
GGGGCAGGC
2148
DRSHLTR
2648
QSGSLTR
3148
RSDHLSR
3648
0.456


 


1071
GGGGCAGGC
2149
DRSHLTR
2649
QSGDLTR
3149
RSDHLSR
3649
0.2


 


1072
GGATTGGCT
2150
QSSDLTR
2650
RSDALTT
3150
QRAHLAR
3650
0.46


 


1073
GGATTGGCT
2151
QSSDLTR
2651
RSDALTK
3151
QRAHLAR
3651
1.37


 


1075
GTGTTGGCG
2152
RSDELTR
2652
RSDALTK
3152
RSDALTR
3652
0.915


 


1076
GCGGCAGCG
2153
RSDELTR
2653
QSGSLTR
3153
RSDERKR
3653
4.1


 


1077
GCGGCAGCG
2154
RSDELTR
2654
QSGDLRR
3154
RSDERKR
3654
6.2


 


1078
GGGGGGGCC
2155
ERGTLAR
2655
RSDHLSR
3155
RSDHLSR
3655
0.2


 


1079
GGGGGGGCC
2156
ERGDLTR
2656
RSDHLSR
3156
RSDHLSR
3656
4.1


 


1080
CTGGAGGCG
2157
RSDELTR
2657
RSDNLAR
3157
RSDALRE
3657
1.37


 


1081
GGGGAGGTG
2158
RSDALTR
2658
RSDNLTR
3158
RSDHLSR
3658
0.05


 


1082
CTGGCGGCG
2159
RSDELTR
2659
RSDELTR
3159
RSDALRE
3659
0.152


 


1083
CTGGTGGCA
2160
QSGDLTR
2660
RSDALSR
3160
RSDALRE
3660
0.152


 


1084
GGTGAGGCG
2161
RSDELTR
2661
RSDNLAR
3161
MSHHLSR
3661
0.5


 


1085
GGTGAGGCG
2162
RSDELTR
2662
RSDNLAR
3162
QSSHLAR
3662
0.46


 


1086
GGGGCTGGG
2163
RSDHLSR
2663
QSSDLQR
3163
RSDHLTR
3663
0.1


 


1087
CGGGCGGCC
2164
ERGDLTR
2664
RSDELQR
3164
RSDHLAE
3664
1.24


 


1088
CGGGCGGCC
2165
ERGDLTR
2665
RSDELQR
3165
RSDHLRE
3665
0.905


 


1089
GACGAGGCT
2166
QSSDLRR
2666
RSDNLAR
3166
DRSNLTR
3666
0.171


 


1090
AAGGCGCTG
2167
RSDALRE
2667
RSDELQR
3167
RSDNLTQ
3667
30.3


 


1091
GTAGAGGAC
2168
DRSNLTR
2668
RSDNLAR
3168
QPASLAR
3668
0.085


 


1092
GCCTTGGCT
2169
QSSDLRR
2669
RGDALTS
3169
DRSDLTR
3669
2.735


 


1093
GCGGAGTCG
2170
RSADLRT
2670
RSDNLAR
3170
RSDERKR
3670
0.046


 


1094
GCGGTTGGT
2171
TSGHLVR
2671
QSSALTR
3171
RSDERKR
3671
12.34


 


1095
GGGGGAGCC
2172
ERGDLTR
2672
QRAHLER
3172
RSDHLSR
3672
0.395


 


1096
GGGGGAGCC
2173
DRSSLTR
2673
QRAHLER
3173
RSDHLSR
3673
0.019


 


1097
GAGGCCGAA
2174
QSANLAR
2674
DCRDLAR
3174
RSDNLAR
3674
0.77


 


1098
GCCGGGGAG
2175
RSDNLTR
2675
RSDHLTR
3175
DRSDLTR
3675
0.055


 


1099
GCGGAGTCG
2176
TSGHLVR
2676
TSGSLTR
3176
RSDERKR
3676
0.45


 


1100
GTGTTGGTA
2177
QSGALTR
2677
RGDALTS
3177
RSDALTR
3677
1.4


 


1101
ATGGGAGTT
2178
TTSALTR
2678
QRAHLER
3178
RSDALRQ
3678
0.065


 


1102
AAGGCAGAA
2179
QSANLAR
2679
QSGSLTR
3179
RSDNLTQ
3679
8.15


 


1103
AAGGCAGAA
2180
QSANLAR
2680
QSGDLTR
3180
RSDNLTQ
3680
1.4


 


1104
CGGGCAGCT
2181
QSSDLRR
2681
QSGSLTR
3181
RSDHLRE
3681
0.08


 


1105
CTGGCAGCC
2182
ERGDLTR
2682
QSGDLTR
3182
RSDALRE
3682
2.45


 


1106
CTGGCAGCC
2183
DRSSLTR
2683
QSGDLTR
3183
RSDALRE
3683
0.19


 


1107
GCGGGAGTT
2184
QSSALAR
2684
QRAHLER
3184
RSDERKR
3684
0.06


 


1108
CAGGCTGGA
2185
QSGHLAR
2685
TSGELVR
3185
RSDNLRE
3685
0.007


 


1109
AGGGGAGCC
2186
ERGDLTR
2686
QRAHLER
3186
RSDHLTQ
3686
0.347


 


1110
AGGGGAGCC
2187
DRSSLTR
2687
QRAHLER
3187
RSDHLTQ
3687
0.095


 


1111
CTGGTAGGG
2188
RSDHLAR
2688
QSSSLVR
3188
RSDALRE
3688
0.095


 


1112
CTGGTAGGG
2189
RSDHLAR
2689
QSATLAR
3189
RSDALRE
3689
0.125


 


1113
CTGGGGGCA
2190
QSGDLTR
2690
RSDHLTR
3190
RSDALRE
3690
0.06


 


1114
CAGGTTGAT
2191
QSSNLAR
2691
TSGSLTR
3191
RSDNLRE
3691
2.75


 


1115
CAGGTTGAT
2192
QSSNLAR
2692
QSSALTR
3192
RSDNLRE
3692
0.7


 


1116
CCGGAAGCG
2193
RSDELTR
2693
QSSNLVR
3193
RSDELRE
3693
12.3


 


1117
GCAGCGCAG
2194
RSSNLRE
2694
RSDELTR
3194
QSGSLTR
3694
2.85


 


1118
TAGGGAGTC
2195
DRSALTR
2695
QRAHLER
3195
RSDNLTT
3695
1.4


 


1119
TGGGAGGGT
2196
TSGHLVR
2696
RSDNLAR
3196
RSDHLTT
3696
0.1


 


1120
AGGGACGCG
2197
RSDELTR
2697
DRSNLTR
3197
RSDHLTQ
3697
2.735


 


1121
CTGGTGGCC
2198
ERGDLTR
2698
RSDALTR
3198
RSDALRE
3698
2.76


 


1122
CTGGTGGCC
2199
DRSSLTR
2699
RSDALTR
3199
RSDALRE
3699
0.101


 


1123
TAGGAAGCA
2200
QSGSLTR
2700
QSGNLAR
3200
RSDNLTT
3700
0.065


 


1124
GTGGATGGA
2201
QSGHLAR
2701
TSGNLVR
3201
RSDALTR
3701
0.101


 


1126
TTGGCTATG
2202
RSDALTS
2702
TSGELVR
3202
RGDALTS
3702
0.46


 


1127
CAGGGGGTT
2203
QSSALAR
2703
RSDHLTR
3203
RSDNLRE
3703
0.1


 


1128
AAGGTCGCC
2204
ERGDLTR
2704
DPGALVR
3204
RSDNLTQ
3704
5.45


 


1130
GGTGCAGAC
2205
DRSNLTR
2705
QSGDLTR
3205
MSHHLSR
3705
0.1


 


1131
GTGGGAGCC
2206
ERGDLTR
2706
QRAHLER
3206
RSDALTR
3706
0.95


 


1132
GGGGCTGGA
2207
QSCHLAR
2707
TSGELVR
3207
RSDHLSR
3707
0.055


 


1133
GGGGCTGGA
2208
QRAHLER
2708
TSGELVR
3208
RSDHLSR
3708
0.5


 


1134
TGGGGGTGG
2209
RSDHLTT
2709
RSDHLTR
3209
RSDHLTT
3709
0.067


 


1135
GCGGCGGGG
2210
RSDHLAR
2710
RSDELQR
3210
RSDERKR
3710
0.025


 


1136
CCGGGAGTG
2211
RSDALTR
2711
QRAHLER
3211
RSDTLRE
3711
0.225


 


1137
CCGGGAGTG
2212
RSSALTR
2712
QRAHLER
3212
RSDTLRE
3712
0.085


 


1138
CAGGGGGTA
2213
QSGALTR
2713
RSDHLTR
3213
RSDNLRE
3713
0.027


 


1139
ACGGCCGAG
2214
RSDNLAR
2714
DRSDLTR
3214
RSDTLTQ
3714
0.535


 


1140
AAGGGTGCG
2215
RSDELTR
2715
QSSHLAR
3215
RSDNLTQ
3715
0.3


 


1141
ATGGACTTG
2216
RGDALTS
2716
DRSNLTR
3216
RSDALTQ
3716
1.7


 


1148
TTGGAGGAG
2217
RSDNLTR
2717
RSDNLTR
3217
RGDALTS
3717
0.006


 


1149
TTGGAGGAG
2218
RSDNLTR
2718
RSDNLTR
3218
RSDALTK
3718
0.004


 


1150
GAAGAGGCA
2219
QSGSLTR
2719
RSDNLTR
3219
QSGNLTR
3719
0.004


 


1151
GTAGTATGG
2220
RSDHLTT
2720
QRSALAR
3220
QRASLAR
3720
1.63


 


1152
AAGGCTGGA
2221
QSGHLAR
2721
TSGELVR
3221
RSDNLTQ
3721
1.605


 


1153
AAGGCTGGA
2222
QRAHLAR
2722
TSGELVR
3222
RSDNLTQ
3722
8.2


 


1154
CTGGCGTAG
2223
RSDNLTT
2723
RSDELQR
3223
RSDALRE
3723
1.04


 


1156
ATGGTTGAA
2224
QSANLAR
2724
QSSALTR
3224
RSDALRQ
3724
7.2


 


1157
ATGGTTGAA
2225
QSANLAR
2725
TSGSLTR
3225
RSDALRQ
3725
0.885


 


1158
AGGGGAGAA
2226
QSANLAR
2726
QSGHLTR
3226
RSDHLTQ
3726
0.1


 


1159
AGGGGAGAA
2227
QSANLAR
2727
QRAHLER
3227
RSDHLTQ
3727
0.555


 


1160
TGGGAAGGC
2228
DRSHLAR
2728
QSSNLVR
3228
RSDHLTT
3728
0.415


 


1161
GAGGCCGCC
2229
DRSHLAR
2729
DRSDLTR
3229
RSDNLAR
3729
0.45


 


1162
GTGTTGGTA
2230
QSGALTR
2730
RADALMV
3230
RSDALTR
3730
0.465


 


1163
GTGTGAGCC
2231
ERGDLTR
2731
QSGHLTT
3231
RSDALTR
3731
1.45


 


1164
GTGTGAGCC
2232
ERGDLTR
2732
QSVHLQS
3232
RSDALTR
3732
15.4


 


1165
GCGAAGGTG
2233
RSDALTR
2733
RSDNLTQ
3233
RSDERKR
3733
1.4


 


1166
GCGAAGGTG
2234
RSDALTR
2734
RSDNLTQ
3234
RSSDRKR
3734
0.195


 


1167
GCGAAGGTG
2235
RSDALTR
2735
RSDNLTQ
3235
RSHDRKR
3735
0.95


 


1168
AAGGCGCTG
2236
RSDALRE
2736
RSSDLTR
3236
RSDNLTQ
3736
2.8


 


1169
GTAGAGGAC
2237
DRSNLTR
2737
RSDNLAR
3237
QSSSLVR
3737
0.053


 


1170
GCCTTGGCT
2238
QSSDLRR
2738
RADALMV
3238
DRSDLTR
3738
2.75


 


1171
GCGGAGTCG
2239
RSDDLRT
2739
RSDNLAR
3239
RSDERKR
3739
0.18


 


1172
GCCGGGGAG
2240
RSDNLTR
2740
RSDHLTR
3240
ERGDLTR
3740
0.01


 


1173
GCTGAAGGG
2241
RSDHLSR
2741
QSGNLAR
3241
QSSDLRR
3741
0.008


 


1174
GCTGAAGGG
2242
RSDHLSR
2742
QSSNLVR
3242
QSSDLRR
3742
0.018


 


1175
AAGGTCGCC
2243
DRSDLTR
2743
DPGALVR
3243
RSDNLTQ
3743
8.9


 


1176
GTGGGAGCC
2244
DRSDLTR
2744
QRAHLER
3244
RSDALTR
3744
4.1


 


1177
CCGGGCGCA
2245
QSGSLTR
2745
DRSHLAR
3245
RSDTLRE
3745
4.1


 


1178
GAGGATGGC
2246
DRSHLAR
2746
TSGNLVR
3246
RSDNLAR
3746
0.085


 


1179
GCAGCGCAG
2247
RSSNLRE
2747
RSSDLTR
3247
QSGSLTR
3747
2.735


 


1180
AAGGAAAGA
2248
QSGHLNQ
2748
QSGNLAR
3248
RSDNLTQ
3748
4.825


 


1181
TTGGCTATG
2249
RSDALRQ
2749
TSGELVR
3249
RGDALTS
3749
8.2


 


1182
CAGGAAGGC
2250
DRSHLAR
2750
QSGNLAR
3250
RSDNLRE
3750
1.48


 


1183
CAGGAAGGC
2251
DRSHLAR
2751
QSSNLVR
3251
RSDNLRE
3751
1.935


 


1184
AAGGAAAGA
2252
KNWKLQA
2752
QSGNLAR
3252
RSDNLTQ
3752
2.785


 


1185
AAGGAAAGA
2253
KNWKLQA
2753
QSHNLAR
3253
RSDNLTQ
3753
5.25


 


1186
GCCGAGGTG
2254
RSDSLLR
2754
RSKNLQR
3254
ERGTLAR
3754
27.5


 


1187
CTGGTGGGC
2255
DRSHLAR
2755
RSDALTR
3255
RSDALRE
3755
0.006


 


1188
GTAGTATGG
2256
RSDHLTT
2756
QSSSLVR
3256
QRASLAR
3756
2.74


 


1189
ATGGTTGAA
2257
QSANLAR
2757
TSGALTR
3257
RSDALRQ
3757
1.51


 


1190
ATGGCAGTG
2258
RSDALTR
2758
QSGDLTR
3258
RSDSLNQ
3758
1.484


 


1191
ATGGCAGTG
2259
RSDALTR
2759
QSGSLTR
3259
RSDSLNQ
3759
5.325


 


1192
ATGGCAGTG
2260
RSDALTR
2760
QSGDLTR
3260
RSDALTQ
3760
2.364


 


1193
ATGGCAGTG
2261
RSDALTR
2761
QSGSLTR
3261
RSDALTQ
3761
3.125


 


1194
GAGAAGGTG
2262
RSDALTR
2762
RSDNRTA
3262
RSDNLTR
3762
2.19


 


1195
GAGAAGGTG
2263
RSDALTR
2763
RSDNRTA
3263
RSSNLTR
3763
2.8


 


1197
GAAGGTGCC
2264
ERCDLTR
2764
MSHHLSR
3264
QSGNLTR
3764
14.8


 


1199
ATGGAGAAG
2265
RSDNRTA
2765
RSDNLTR
3265
RSDALTQ
3765
3.428


 


1200
ATGCAGAAG
2266
RSDNRTA
2766
RSSNLTR
3266
RSDALTQ
3766
16.87


 


1201
ATGGAGAAG
2267
RSDNRTA
2767
RSHNLTR
3267
RSDALTQ
3767
14.8


 


1202
CTGGAGTAC
2268
DRSNLRT
2768
RSDNLTR
3268
RSDALRE
3768
2.834


 


1203
GGAGTACTG
2269
RSDALRE
2769
QRSALAR
3269
QRAHLAR
3769
2.945


 


1204
GGAGTACTG
2270
RSDALRE
2770
QSSSLVR
3270
QRAHLAR
3770
4.38


 


1205
CGGGCAGCT
2271
QSSDLRR
2771
QSGDLTR
3271
RSDHLRE
3771
0.9


 


1206
GCGGGAGTT
2272
TTSALTR
2772
QRAHLER
3272
RSDERKR
3772
0.034


 


1207
CAGGCTGGA
2273
QRAHLER
2773
TSGELVR
3273
RSDNLRE
3773
0.45


 


1209
CCGGAAGCG
2274
RSDELTR
2774
QSSNLVR
3274
RSDTLRE
3774
19.28


 


1211
GCAGCGCAG
2275
RSDNLRE
2775
RSDELTR
3275
QSGSLTR
3775
6.5


 


1212
CAGGGGGTT
2276
TTSALTR
2776
RSDHLTR
3276
RSDNLRE
3776
0.05


 


1213
GAAGAAGAG
2277
RSDNLTR
2777
QSSNLVR
3277
QSGNLTR
3777
12.3


 


1214
ATGGGAGTT
2278
TTSALTR
2778
QRAHLER
3278
RSDALTQ
3778
0.46


 


1215
GTGGGGGCT
2279
QSSDLRR
2779
RSDHLTR
3279
RSDALTR
3779
0.003


 


1217
GAAGAGGCA
2280
QSGSLTR
2780
RSDNLTR
3280
QSANLTR
3780
0.004


 


1218
GCGGTGAGG
2281
RSDHLTQ
2781
RSQALTR
3281
RSDERKR
3781
0.46


 


1219
AAGGAAAGG
2282
RSDHLTQ
2782
QSHNLAR
3282
RSDNLTQ
3782
0.68


 


1220
AAGGAAAGG
2283
RSDHLTQ
2783
QSGNLAR
3283
RSDNLTQ
3783
0.175


 


1221
AAGGAAAGG
2284
RSDHLTQ
2784
QSSNLVR
3284
RSDNLTQ
3784
1.4


 


1222
CAGGAGGGC
2285
DRSHLAR
2785
RSDNLAR
3285
RSDNLRE
3785
0.155


 


1223
ATGGACTTG
2286
RSDALTK
2786
DRSNLTR
3286
RSDALTQ
3786
7


 


1224
ATGGACTTG
2287
RADALMV
2787
DRSNLTR
3287
RSDALTQ
3787
12


 


1227
GAATAGGGG
2288
RSDHLSR
2788
RSDHLTK
3288
QSGNLAR
3788
25


 


1228
ACGGCCGAG
2289
RSDNLAR
2789
DRSDLTR
3289
RSDDLTQ
3789
12


 


1229
AAGGGTGCG
2290
RSDELTR
2790
MSHHLSR
3290
RSDNLTQ
3790
8.2


 


1230
AAGGGAGAC
2291
DRSNLTR
2791
QSGHLTR
3291
RSDNLTQ
3791
0.383


 


1231
AAGGGAGAC
2292
DRSNLTR
2792
QRAHLER
3292
RSDNLTQ
3792
0.213


 


1232
TGGGACCTG
2293
RSDALRE
2793
DRSNLTR
3293
RSDHLTT
3793
0.113


 


1233
TGGGACCTG
2294
RSDALRE
2794
DRSNLTR
3294
RSDHLTT
3794
0.635


 


1234
GAGTAGGCA
2295
QSGSLTR
2795
RSDNLTK
3295
RSDNLAR
3795
0.101


 


1236
GAGTAGGCA
2296
QSGSLTR
2796
RSDHLTT
3296
RSDNLAR
3796
0.065


 


1237
GAAGGAGAG
2297
RSDNLAR
2797
QRAHLER
3297
QSGNLAR
3797
0.065


 


1238
CTGGATGTT
2298
QSSALAR
2798
TSGNLVR
3298
RSDALRE
3798
0.313


 


1239
CAGGACGTG
2299
RSDALTR
2799
DPGNLVR
3299
RSDNLKD
3799
0.144


 


1240
GGGGAGGCA
2300
QSGSLTR
2800
RSDNLTR
3300
RSDHLSR
3800
0.056


 


1241
GAGGTGTCA
2301
QSHDLTK
2801
RSDALAR
3301
RSDNLAR
3801
0.027


 


1242
GGGGTTGAA
2302
QSMJLAR
2802
TSGSLTR
3302
RSDHLSR
3802
0.02


 


1243
GGGGTTGAA
2303
QSANLAR
2803
QSSALTR
3303
RSDHLSR
3803
0.101


 


1244
GTCGCGGTG
2304
RSDALTR
2804
RSDELQR
3304
DRSALAR
3804
0.044


 


1245
GTCGCGGTG
2305
RSDALTR
2805
RSDELQR
3305
DSGSLTR
3805
0.102


 


1246
GTGGTTGCG
2306
RSDELTR
2806
TSGSLTR
3306
RSDALTR
3806
0.051


 


1247
GTGGTTGCG
2307
RSDELTR
2807
TSGALTR
3307
RSDALTR
3807
0.117


 


1248
GTCTAGGTA
2308
QSGALTR
2808
RSDNLTT
3308
DRSALAR
3808
5.14


 


1249
CCGGGAGCG
2309
RSDELTR
2809
QSGHLTR
3309
RSDTLRE
3809
0.26


 


1250
GAAGGAGAG
2310
RSDNLAR
2810
QSGHLTR
3310
QSGNLAR
3810
0.31


 


1252
CCGGCTGGA
2311
QRAHLER
2811
QSSDLTR
3311
RSDTLRE
3811
0.153


 


1253
CCGGGAGCG
2312
RSDELTR
2812
QRAHLER
3312
RSDTLRE
3812
0.228


 


1255
ACGTAGTAG
2313
RSDNLTT
2813
RSDNLTK
3313
RSDTLKQ
3813
0.69


 


1256
GGGGAGGAT
2314
QSSNLAR
2814
RSDNLQR
3314
RSDHLSR
3814
2


 


1257
GGGGAGGAT
2315
TTSNLAR
2815
RSDNLQR
3315
RSDHLSR
3815
1


 


1258
GGGGAGGAT
2316
QSSNLRR
2816
RSDNLQR
3316
RSDHLSR
3816
2


 


1259
GAGTGTGTG
2317
RSDSLLR
2817
DRDHLTR
3317
RSDNLAR
3817
1.5


 


1260
GAGTGTGTG
2318
RLDSLLR
2818
DRDHLTR
3318
RSDNLAR
3818
1.8


 


1261
TGCGGGGCA
2319
QSGDLTR
2819
RSDHLTR
3319
RRDTLHR
3819
0.2


 


1262
TGCGGGGCA
2320
QSGDLTR
2820
RSDHLTR
3320
RLDTLGR
3820
3


 


1263
TGCGGGGCA
2321
QSGDLTR
2821
RSDHLTR
3321
DSGHLAS
3821
21


 


1264
AAGTTGGTT
2322
TTSALTR
2822
RADALMV
3322
RSDNLTQ
3822
0.21


 


1265
AAGTTGGTT
2323
TTSALTR
2823
RSDALTT
3323
RSDNLTQ
3823
0.077


 


1266
CAGGGTGGC
2324
DRSHLTR
2824
QSSHLAR
3324
RSDNLRE
3824
6.1


 


1267
TAGGCAGTC
2325
DRSALTR
2825
QSGSLTR
3325
RSDNLTT
3825
6


 


1268
CTGTTGGCT
2326
QSSDLTR
2826
RADALMV
3326
RSDALRE
3826
1.52


 


1269
CTGTTGGCT
2327
QSSDLTR
2827
RSDALTT
3327
RSDALRE
3827
12.3


 


1270
TTGGATGGA
2328
QSGHLAR
2828
TSGNLVR
3328
RSDALTK
3828
0.4


 


1271
GTGGCACTG
2329
RSDALRE
2829
QSGSLTR
3329
RSDALTR
3829
0.915


 


1272
CAGGAGTCC
2330
DRSSLTT
2830
RSDNLAR
3330
RSDNLRE
3830
0.04


 


1273
CAGGAGTCC
2331
ERGDLTT
2831
RSDNLAR
3331
RSDNLRE
3831
0.1


 


1274
GCATGGGAA
2332
QSANLSR
2832
RSDHLTT
3332
QSGSLTR
3832
0.306


 


1275
GCATGGGAA
2333
QRSNLVR
2833
RSDHLTT
3333
QSGSLTR
3833
0.326


 


1276
TAGGAAGAG
2334
RSDNLAR
2834
QRSNLVR
3334
RSDNLTT
3834
0.685


 


1277
GAAGAGGGG
2335
RSDHLAR
2835
RSDNLAR
3335
QSGNLTR
3835
0.421


 


1278
GAGTAGGCA
2336
QSGSLTR
2836
RSDNLRT
3336
RSDNLAR
3836
0.019


 


1279
GAGGTGTCA
2337
QSGDLRT
2837
RSDALAR
3337
RSDNLAR
3837
0.025


 


1282
TCGGTCGCC
2338
ERGDLTR
2838
DPGALVR
3338
RSDELRT
3838
74.1


 


1287
GTGGTAGGA
2339
QSGHLAR
2839
QSGALAR
3339
RSDALTR
3839
0.152


 


1288
CAGGGTGGC
2340
DRSHLTR
2840
QSSHLAR
3340
RSDNLTE
3840
4.1


 


1289
TAGGCAGTC
2341
DRSALTR
2841
QSGSLTR
3341
RSDNLTK
3841
1.37


 


1290
GTGGTGATA
2342
QSGALTQ
2842
RSHALTR
3342
RSDALTR
3842
24.05


 


1291
GTGGTGATA
2343
QQASLNA
2843
RSHALTR
3343
RSDALTR
3843
20.55


 


1292
TTGGATGGA
2344
QSGHLAR
2844
TSGNLVR
3344
RSDALTT
3844
4.12


 


1293
AAGGTAGGT
2345
TSGHLVR
2845
QSGALAR
3345
RSDNLTQ
3845
0.457


 


1294
AAGGTAGGT
2346
MSHHLSR
2846
QSGALAR
3346
RSDNLTQ
3846
2.75


 


1295
CAGGAGTCC
2347
DRSSLTT
2847
RSDNLAR
3347
RSDNLTE
3847
0.116


 


1296
CAGGAGTCC
2348
ERGDLTT
2848
RSDNLAR
3348
RSDNLTE
3848
37


 


1297
TAGGAAGAG
2349
RSDNLAR
2849
QRSNLVR
3349
RSDNLTK
3849
0.05


 


1298
CAGGACGTG
2350
RSDLATR
2850
DPGNLVR
3350
RSDNLTE
3850
0.05


 


1300
GTCTAGGTA
2351
QSGALTR
2851
RSDNLTK
3351
DRSALAR
3851
0.46


 


1302
CCGGCTGGA
2352
QSGHLTR
2852
QSSDLTR
3352
RSDTLRE
3852
0.05


 


1303
TAGGAGTTT
2353
QRSALAS
2853
RSDNLAR
3353
RSDNLTT
3853
0.088


 


1306
CTGGCCTTG
2354
RSDALTT
2854
DCRDLAR
3354
RSDALRE
3854
2.285


 


1308
TGGGCAGCC
2355
ERGTLAR
2855
QSGSLTR
3355
RSDHLTT
3855
0.305


 


1309
TAGGAGTTT
2356
QSSALAS
2856
RSDNLAR
3356
RSDNLTT
3856
0.184


 


1310
TAGGAGTTT
2357
TTSALAS
2857
RSDNLAR
3357
RSDNLTT
3857
0.075


 


1311
TGGGCAGCC
2358
ERGDLAR
2858
QSGSLTR
3358
RSDHLTT
3858
0.91


 


1312
GGGGCGTGA
2359
QSGHLTK
2859
RSDELQR
3359
RSDHLSR
3859
0.23


 


1313
GGGGCGTGA
2360
QSCHLTT
2860
RSDELQR
3360
RSDHLSR
3860
0.09


 


1314
GTACAGTAG
2361
RSDNLTT
2861
RSDNLRE
3361
QSSSLVR
3861
3.09


 


1315
GTACAGTAG
2362
RSDNLTT
2862
RSDNLTE
3362
QSSSLVR
3862
9.27


 


1318
ATGGTGTGT
2363
TSSHLAS
2863
RSDALAR
3363
RSDALAQ
3863
0.048


 


1319
ATGGTGTGT
2364
MSHHLTT
2864
RSDALAR
3364
RSDALAQ
3864
0.228


 


1320
TTGGGAGAG
2365
RSDNLAR
2865
QPAHLER
3365
RSDALTT
3865
0.044


 


1321
TTGGGAGAG
2366
RSDNLAR
2866
QRAHLER
3366
RADALMV
3866
0.127


 


1322
GTGGGAATA
2367
QSGALTQ
2867
QSGHLTR
3367
RSDALTR
3867
0.799


 


1323
GTGGGAATA
2368
QLTGLNQ
2868
QSGHLTR
3368
RSDALTR
3868
0.744


 


1324
GTGGGAATA
2369
QQASLNA
2869
QSHHLTR
3369
RSDALTR
3869
18.52


 


1325
TTGGTTGGT
2370
TSGHLVR
2870
TSGSLTR
3370
RSDALTK
3870
0.306


 


1326
TTGGTTGGT
2371
TSGHLVR
2871
QSSALTR
3371
RSDALTK
3871
4.385


 


1327
TTGGTTGGT
2372
TSGHLVR
2872
TSGSLTR
3372
RSDALTT
3872
0.566


 


1328
TTGGTTGGT
2373
TSGHLVR
2873
QSSALTR
3373
RSDALTT
3873
7.95


 


1329
CTGGCCTGG
2374
RSDHLTT
2874
DRSDLTR
3374
RSDALRE
3874
0.68


 


1330
GAGGTGTGA
2375
QSGHLTT
2875
RSDALTR
3375
RSDNLAR
3875
0.175


 


1331
CTGGCCTGG
2376
RSDHLTT
2876
DCRDLAR
3376
RSDALRE
3876
0.388


 


1334
CCGGCGCTG
2377
RSDALRE
2877
RSSDLTR
3377
RSDDLRE
3877
0.31


 


1335
GACGCTGGC
2378
DRSHLTR
2878
QSSDLTR
3378
DSSNLTR
3878
1.4


 


1336
CGGGCTGGA
2379
QSGHLAR
2879
QSSDLTR
3379
RSDHLAE
3879
1.4


 


1337
CGGGCTGGA
2380
QSSHLAR
2880
QSSDLTR
3380
RSDHLAE
3880
0.235


 


1338
GGGATGGCG
2381
RSDELTR
2881
RSDALTQ
3381
RSDHLSR
3881
1.04


 


1339
GGGATGGCG
2382
RSDELTR
2882
RSDSLTQ
3382
RSDHLSR
3882
0.569


 


1340
GGGATGGCG
2383
RSDELTR
2883
RSDALTQ
3383
RSHHLSR
3883
0.751


 


1341
GGGATGGCG
2384
RSDELTR
2884
RSDSLTQ
3384
RSHHLSR
3884
4.1


 


1342
CAGGCGCAG
2385
RSDNLRE
2885
RSSDLTR
3385
RSDNLTE
3885
0.68


 


1343
CAGGCGCAG
2386
RSDNLTT
2886
RTSTLTR
3386
RSDNLTE
3886
37.04


 


1344
CCGGGCGAC
2387
DRSNLTR
2887
DRSHLAR
3387
RSDTLRE
3887
2.28


 


1346
GATGTGTGA
2388
QSGHLTT
2888
RSDALAR
3388
TSANLSR
3888
0.153


 


1347
CAGTGAATG
2389
RSDALTS
2889
QSHHLTT
3389
RSDNLTE
3889
8.23


 


1348
GGGTCACTG
2390
RSDALTA
2890
QAATLTT
3390
RSDHLSR
3890
2.58


 


1350
CAGTGAATG
2391
RSDALTQ
2891
QSGHLTT
3391
RSDNLTE
3891
74.1


 


1351
GGGTCACTG
2392
RSDALRE
2892
QSHDLTK
3392
RSDHLSR
3892
0.234


 


1352
GTGTGGGTC
2393
DRSALAR
2893
RSDHLTT
3393
RSDALTR
3893
0.023


 


1353
CTGGCGAGA
2394
QSGHLNQ
2894
RSDELQR
3394
RSDALRE
3894
56.53


 


1354
CTGGCGAGA
2395
KNWKLQA
2895
RSDELQR
3395
RSDALRE
3895
20.85


 


1355
GCTTTGGCA
2396
QSGSLTR
2896
RSDALTT
3396
QSSDLTR
3896
0.172


 


1356
GCTTTGGCA
2397
QSGSLTR
2897
RADALMV
3397
QSSDLTR
3897
0.034


 


1357
GACTTGGTA
2398
QSSSLVR
2898
RSDALTT
3398
DRSNLTR
3898
0.032


 


1358
GACTTGGTA
2399
QSSSLVR
2899
RADALMV
3399
DRSNLTR
3899
0.05


 


1360
CAGTTGTGA
2400
QSGHLTT
2900
RADALMV
3400
RSDNLTE
3900
41.7


 


1361
AAGGAAAAA
2401
QKTNLDT
2901
QSGNLQR
3401
RSDNLTQ
3901
0.835


 


1362
AAGGAAAAA
2402
QSGNLNQ
2902
QSGNLQR
3402
RSDNLTQ
3902
0.332


 


1363
AAGGAAAAA
2403
QKTNLDT
2903
QRSNLVR
3403
RSDNLTQ
3903
74.1


 


1364
ATGGGTGAA
2404
QSANLSR
2904
QSSHLAR
3404
RSDALAQ
3904
1.22


 


1365
ATGGGTGAA
2405
QRSNLVR
2905
QSSHLAR
3405
RSDALAQ
3905
0.152


 


1366
ATGGGTGAA
2406
QSANLSR
2906
TSGHLVR
3406
RSDALAQ
3906
22.63


 


1367
ATGGGTGAA
2407
QRSNLVR
2907
TSGHLVR
3407
RSDALAQ
3907
1.028


 


1368
CTGGGAGAT
2408
QSSNLAR
2908
QRAHLER
3408
RSDALRE
3908
0.051


 


1369
CTGGGAGAT
2409
QSSNLAR
2909
QSGHLTR
3409
RSDALRE
3909
0.227


 


1373
GTGGTGGGC
2410
DRSHLTR
2910
RSDALSR
3410
RSDALTR
3910
0.025


 


1374
CCGGCGGTG
2411
RSDALTR
2911
RSDELQR
3411
RSDELRE
3911
0.003


 


1375
CCGGCGGTG
2412
RSDALTR
2912
RSDDLQR
3412
RSDELRE
3912
0.008


 


1376
CCGGCGGTG
2413
RSDALTR
2913
RSDERKR
3413
RSDELRE
3913
0.858


 


1377
CCGGCGGTG
2414
RSDALTR
2914
RSDELQR
3414
RSDDLRE
3914
0.012


 


1378
CCGGCGGTG
2415
RSDALTR
2915
RSDDLQR
3415
RSDDLRE
3915
0.012


 


1379
CCGGCGGTG
2416
RSDALTR
2916
RSDERKR
3416
RSDDLRE
3916
0.25


 


1380
GCCGACGGT
2417
QSSHLTR
2917
DRSNLTR
3417
ERGDLTR
3917
0.076


 


1381
GCCGACGGT
2418
QSSHLTR
2918
DPGNLVR
3418
ERGDLTR
3918
0.23


 


1382
GCCGACGGT
2419
QSSHLTR
2919
DRSNLTR
3419
DCRDLAR
3919
3.1


 


1383
GCCGACGGT
2420
QSSHLTR
2920
DPGNLVR
3420
DGRDLAR
3920
1.74


 


1384
GGTGTGGGC
2421
DRSHLTR
2921
RSDALSR
3421
MSHHLSR
3921
0.013


 


1385
TGGGCAAGA
2422
QSGHLNQ
2922
QSGSLTR
3422
RSDHLTT
3922
0.229


 


1386
TGGGCAAGA
2423
ENWKLQA
2923
QSGSLTR
3423
RSDHLTT
3923
0.193


 


1389
CTGGCCTGG
2424
RSDHLTT
2924
DCRDLAR
3424
RSDALRE
3924
0.175


 


1393
TGGGAAGCT
2425
QSSDLRR
2925
QSGNLAR
3425
RSDHLTT
3925
0.1


 


1394
TGGGAAGCT
2426
QSSDLRR
2926
QSGNLAR
3426
RSDHLTK
3926
0.04


 


1395
GAAGAGGGA
2427
QSGHLQR
2927
RSDNLAR
3427
QSGNLAR
3927
0.025


 


1396
GAAGAGGGA
2428
QRAHLAR
2928
RSDNLAR
3428
QSGNLAR
3928
0.107


 


1397
GAAGAGGGA
2429
QSSHLAR
2929
RSDNLAR
3429
QSGNLAR
3929
0.14


 


1398
TAATGGGGG
2430
RSDHLSR
2930
RSDHLTT
3430
QSGNLRT
3930
0.065


 


1399
TGGGAGTGT
2431
TKQHLKT
2931
RSDNLAR
3431
RSDHLTT
3931
0.1


 


1400
CCGGGTGAG
2432
RSDNLAR
2932
QSSHLAR
3432
RSDDLRE
3932
0.371


 


1401
GAGTTGGCC
2433
ERGTLAR
2933
RADALMV
3433
RSDNLAR
3933
0.167


 


1402
CTGGAGTTG
2434
RGDALTS
2934
RSDNLAR
3434
RSDALRE
3934
0.15


 


1403
ATGGCAATG
2435
RSDALTQ
2935
QSGSLTR
3435
RSDALTQ
3935
0.07


 


1404
GAGGCAGGG
2436
RSDHLSR
2936
QSGSLTR
3436
RSDNLAR
3936
0.022


 


1405
GAGGCAGGG
2437
RSDHLSR
2937
QSGDLTR
3437
RSDNLAR
3937
0.045


 


1406
GAAGCGGAG
2438
RSDNLAR
2938
RSDELTR
3438
QSGNLAR
3938
0.025


 


1407
GCGGGCGCA
2439
QSGSLTR
2939
DRSHLAR
3439
RSDERKR
3939
0.585


 


1408
CCGGCAGGG
2440
RSDHLSR
2940
QSGSLTR
3440
RSDELRE
3940
0.305


 


1409
CCGGCAGGG
2441
RSDHLSR
2941
QSGSLTR
3441
RSDDLRE
3941
0.153


 


1410
CCGGCGGCG
2442
RSDELTR
2942
RSDELQR
3442
RSDELRE
3942
0.814


 


1411
TGAGGCGAG
2443
RSDNLAR
2943
DRSHLAR
3443
QSGHLTK
3943
0.282


 


1412
CTGGCCGTG
2444
RSDSLLR
2944
ERGTLAR
3444
RSDALRE
3944
0.172


 


1413
CTGGCCGCG
2445
RSDELTR
2945
DRSDLTR
3445
RSDALRE
3945
0.152


 


1414
CTGGCCGCG
2446
RSDELTR
2946
ERGTLAR
3446
RSDALRE
3946
0.914


 


1415
GCGGCCGAG
2447
RSDNLAR
2947
DRSDLTR
3447
RSDELQR
3947
0.102


 


1416
GCGGCCGAG
2448
RSDNLAR
2948
ERGTLAR
3448
RSDELQR
3948
0.153


 


1417
GAGTTGGCC
2449
ERGTLAR
2949
RGDALTS
3449
RSDNLAR
3949
1.397


 


1418
CTGGAGTTG
2450
RADALMV
2950
RSDNLAR
3450
RSDALRE
3950
0.241


 


1422
GGGTCGGCG
2451
RSDELTR
2951
RSDDLTT
3451
RSDHLSR
3951
0.064


 


1423
GGGTCGGCG
2452
RSDELTR
2952
RSDDLTK
3452
RSDHLSR
3952
0.034


 


1424
CAGGGCCCG
2453
RSDELRE
2953
DRSHLAR
3453
RSDNLRE
3953
1.37


 


1427
CAGGGCCCG
2454
RSDDLRE
2954
DRSHLAR
3454
RSDNLTE
3954
0.271


 


1428
TGAGGCGAG
2455
RSDNLAR
2955
DRSHLAR
3455
QSVHLQS
3955
0.102


 


1429
TGAGGCGAG
2456
RSDNLAR
2956
DRSHLAR
3456
QSGHLTT
3956
0.074


 


1430
TCGGCCGCC
2457
ERGTLAR
2957
DRSDLTR
3457
RSDDLTK
3957
0.352


 


1431
TCGGCCGCC
2458
ERGTLAR
2958
DRSDLTR
3458
RSDDLAS
3958
6.17


 


1432
TCGGCCGCC
2459
ERGTLAR
2959
ERGTLAR
3459
RSDDLTK
3959
1.778


 


1434
CTGGCCGTG
2460
RSDSLLR
2960
DRSDLTR
3460
RSDALRE
3960
0.051


 


1435
TAATGGGGG
2461
RSDHLSR
2961
RSDHLTT
3461
QSGNLTK
3961
0.057


 


1436
TGGGAGTGT
2462
TSDHLAS
2962
RSDNLAR
3462
RSDHLTT
3962
0.026


 


1439
GGAGTGTTA
2463
QRSALAS
2963
RSDALAR
3463
QSGHLQR
3963
0.075


 


1440
GGAGTGTTA
2464
QSGALTK
2964
RSDALAR
3464
QSGHLQR
3964
0.035


 


1441
ATAGCTGGG
2465
RSDHLSR
2965
QSSDLTR
3465
QSGALTQ
3965
0.262


 


1442
TGCTGGGCC
2466
ERGTLAR
2966
RSDHLTT
3466
DRSHLTK
3966
0.36


 


1443
TGGAAGGAA
2467
QSGNLAR
2967
RSDNLTQ
3467
RSHHLTT
3967
0.22


 


1444
TGGAAGGAA
2468
QSGNLAR
2968
RSDNLTQ
3468
RSSHLTT
3968
0.09


 


1445
TGGAAGCAA
2469
QSGNLAR
2969
RLDNLTA
3469
RSHHLTT
3969
0.182


 


1446
TGGAAGGAA
2470
QSGNLAR
2970
RLDNLTA
3470
RSSHLTT
3970
0.42


 


1454
GGAGAGGCT
2471
QSSDLRR
2971
RSDNLAR
3471
QSGHLQR
3971
0.01


 


1455
CGGGATGAA
2472
QSANLSR
2972
TSGNLVR
3472
RSDHLRE
3972
0.043


 


1456
GGAGAGGCT
2473
QSSDLRR
2973
RSDNLAR
3473
QRAHLAR
3973
0.016


 


1457
GCAGAGGAA
2474
QSANLSR
2974
RSDNLAR
3474
QSGSLTR
3974
0.014


 


1460
TTGGGGGAG
2475
RSDNLAR
2975
RSDHLTR
3475
RADALMV
3975
0.007


 


1461
GACGAGGAG
2476
RSANLAR
2976
RSDNLTR
3476
DRSNLTR
3976
0.014


 


1462
CGGGATGAA
2477
QSGNLAR
2977
TSGNLVR
3477
RSDHLRE
3977
0.05


 


1463
GAGGCTGTT
2478
TTSALTR
2978
QSSDLTR
3478
RSDNLAR
3978
0.003


 


1464
GACGAGGAG
2479
RSDNLAR
2979
RSDNLTR
3479
DRSNLTR
3979
0.002


 


1465
CTGGGAGTT
2480
TTSALTR
2980
QSGHLQR
3480
RSDALRE
3980
0.018


 


1466
CTGGGAGTT
2481
NRATLAR
2981
QSGHLQR
3481
RSDALRE
3981
0.017


 


1468
GGTGATGTC
2482
DRSALTR
2982
TSGNLVR
3482
MSHHLSR
3982
0.08


 


1469
GGTGATGTC
2483
DRSALTR
2983
TSGNLVR
3483
TSGHLVR
3983
0.28


 


1470
GGTGATGTC
2484
DRSALTR
2984
TSGNLVR
3484
QRAHLER
3984
0.156


 


1471
CTGGTTGGG
2485
RSDHLSR
2985
QSSALTR
3485
RSDALRE
3985
0.09


 


1472
TTGAAGGTT
2486
TTSALTR
2986
RSDNLTQ
3486
RADALMV
3986
3.22


 


1473
TTGAAGGTT
2487
TTSALTR
2987
RSDNLTQ
3487
RSDSLTT
3987
0.47


 


1474
TTGAAGGTT
2488
QSSALAR
2988
RSDNLTQ
3488
RADALMV
3988
1.39


 


1475
TTGAAGGTT
2489
QSSALAR
2989
RSDNLTQ
3489
RLHSLTT
3989
0.39


 


1476
TTGAAGGTT
2490
QSSALAR
2990
RSDNLTQ
3490
RSDSLTT
3990
0.305


 


1477
GCAGCCCGG
2491
RSDHLRE
2991
DRSDLTR
3491
QSGSLTR
3991
2.31


 


1479
GAAAGTTCA
2492
QSHDLTK
2992
MSHHLTQ
3492
QSGNLAR
3992
37.04


 


1480
GAAAGTTCA
2493
NKTDLGK
2993
TSGHLVQ
3493
QSGNLAR
3993
62.5


 


1481
GAAAGTTCA
2494
NKTDLGK
2994
TSDHLAS
3494
RSDELRE
3994
37.04


 


1482
CCGTGTGAC
2495
DRSNLTR
2995
TSDHLAS
3495
RSDELRE
3995
111.1


 


1483
CCGTGTGAC
2496
DRSNLTR
2996
MSHHLTT
3496
RSDELRE
3996
20.8


 


1484
GAAGTGGTA
2497
QSSSLVR
2997
RSDALSR
3497
QSGNLAR
3997
0.01


 


1485
AAGTGAGCT
2498
QSSDLRR
2998
QSGHLTT
3498
RSDNLTQ
3998
1.537


 


1486
GGGTTTGAC
2499
DRSNLTR
2999
TTSALAS
3499
RSDHLSR
3999
0.085


 


1487
TTGAAGGTT
2500
TTSALTR
3000
RSDNLTQ
3500
RLHSLTT
4000
0.188


 


1488
AAGTGGTAG
2501
QSSDLRR
3001
QSGHLTT
3501
RLDNRTQ
4001
5.64


 


1490
CTGGTTGGC
2502
RSDHLSR
3002
TSGSLTR
3502
RSDALRE
4002
0.04


 


1491
AAGGGTTCA
2503
NKTDLGK
3003
DSSKLSR
3503
RLDNRTA
4003
4.12


 


1492
AAGTGGTAG
2504
RSDNLTT
3004
RSDHLTT
3504
RSDNLTQ
4004
1.37


 


1493
AAGTGGTAC
2505
RSDNLTT
3005
RSDHLTT
3505
RLDNRTQ
4005
15.09


 


1494
GGGTTTGAC
2506
DRSNLTR
3006
QRSALAS
3506
RSDHLSR
4006
0.255


 


1496
TTGGGGGAG
2507
RSDNLAR
3007
RSDHLTR
3507
RSDALTT
4007
0.065


 


1497
GAGGCTCTT
2508
QSSALAR
3008
QSSDLTR
3508
RSDNLAR
4008
0.007


 


1498
GAGGTTGAT
2509
QSSNLAR
3009
QSSALTR
3509
RSDNLAR
4009
0.101


 


1499
GAGGTTGAT
2510
QSSNLAR
3010
TSGALTR
3510
RSDNLAR
4010
0.02


 


1500
GCAGAGGAA
2511
QSGNLAR
3011
RSDNLAR
3511
QSGSLTR
4011
0.003


 


1522
GCAATGGGT
2512
TSGHLVR
3012
RSDALTQ
3512
QSGDLTR
4012
0.08



















TABLE 6









FINGER (N → C)

















SEQ

SEQ

SEQ



TRIPLET

ID

ID

ID


(5′→3′)
F1
NO:
F2
NO:
F3
NO:





AGG




RXDHXXQ
4055



 


ATG




RXDAXXQ
4056


 


CGG




RXDHXXE
4057


 


GAA


QXGNXXR
4058


 


GAC
DXSNXXR
4059


DXSNXXR
4059


 


GAG
RXDNXXR
4060
RXSNXXR
4061
RXDNXXR
4060


 





RXDNXXR
4060


 


GAT
QXSNXXR
4062
TXGNXXR
4064


 



TXSNXXR
4063


 



TXGNXXR
4064


 


GCA
QXGSXXR
4065
QXGDXXR
4066


 


GCC
EXGTXXR
4067


 


GCG
RXDEXXR
4068
RXDEXXR
4068
RXDEXXR
4068


 







RXDTXXK
4069


 


GCT
QXSDXXR
4070
TXGEXXR
4071


 





QXSDXXR
4070


 


GGA


QXGHXXR
4072
QXAHXXR
4073


 


GGC
DXSHXXR
4074
DXSHXXR
4074


 


GGG
RXDHXXR
4075
RXDHXXR
4075
RXDHXXR
4075


 







RXDHXXK
4076


 


GGT




TXGHXXR
4077


 


GTA


QXGSXXR
4065


 





QXATXXR
4078


 


GTG
RXDAXXR
4079
RXDAXXR
4079
RXDAXXR
4079


 



RXDSXXR
4080


 


TAG


RXDNXXT
4081


 


TCG
RXDDXXK
4082


 


TGT


TXDHXXS
4083

## Claims

1. A method for modulating expression of a gene in a cell, wherein the gene comprises a target site, wherein the target site comprises, in a 3′ to 5′ direction, a first (S1), a second (S2) and a third (S3) target subsites, each target subsite having the nucleotide sequence GNN, the method comprising:
introducing a polynucleotide encoding a zinc finger protein, into the cell, wherein:
(a) the zinc finger protein is a non-naturally-occurring zinc finger protein that has been engineered to bind the target site; and
(b) the zinc finger protein comprises first (F1), second (F2), and third (F3) zinc fingers, ordered F1, F2, F3 from N-terminus to C-terminus that bind to (S1), (S2), and (S3), wherein if S1 comprises GAA, F1 comprises the amino acid sequence QRSNLVR (SEQ ID NO:158); if S1 comprises GAG, F1 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S1 comprises GAC, F1 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S1 comprises GAT, F1 comprises the amino acid sequence QSSNLAR (SEQ ID NO:1765); if S1 comprises GGA, F1 comprises the amino acid sequence QSGHLAR (SEQ ID NO:413); if S1 comprises GGG, F1 comprises the amino acid sequence RSDHLAR (SEQ ID NO:127); if S1 comprises GGC, F1 comprises the amino acid sequence DRSHLTR (SEQ ID NO:1506); if S1 comprises GGT, F1 comprises the amino acid sequence QSSHLTR (SEQ ID NO:835); if S1 comprises GCA, F1 comprises QSGSLTR (SEQ ID NO:342); if S1 comprises GCG, F1 comprises RSDDLTR (SEQ ID NO:188); if S1 comprises GCC, F1 comprises ERGTLAR (SEQ ID NO:131); if S1 comprises GCT, F1 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S1 comprises GTA, F1 comprises the amino acid sequence QSGALTR (SEQ ID NO:1398); if S1 comprises GTG, F1 comprises the amino acid sequence RSDALTR (SEQ ID NO:153); if S1 comprises GTC, F1 comprises the amino acid sequence DRSALAR (SEQ ID NO:184); selecting the F2 zinc finger such that it binds to the S2 target subsite, wherein S2 comprises GAA, F2 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S2 comprises GAG, F2 comprises the amino acid sequence RSDNLAR (SEQ ID NO:130); if S2 comprises GAC, F2 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S2 comprises GAT, F2 comprises the amino acid sequence TSGNLVR (SEQ ID NO:1442); if S2 comprises GGA, F2 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S2 comprises GGG, F2 comprises the amino acid sequence RSDHLSR (SEQ ID NO:229); if S2 comprises GGC, F2 comprises the amino acid sequence DRSHLAR (SEQ ID NO:1092); if S2 comprises GGT, F2 comprises the amino acid sequence TSGHLSR (SEQ ID NO:1201); if S2 comprises GCA, F2 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S2 comprises GCG, F2 comprises the amino acid sequence RSDDLQR (SEQ ID NO:1844); if S2 comprises GCC, F2 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S2 comprises GCT, F2 comprises the amino acid sequence QSSDLTR (SEQ ID NO:1450); if S2 comprises GTA, F2 comprises the amino acid sequence QSGALAR (SEQ ID NO:3339); if S2 comprises GTG, F2 comprises the amino acid sequence RSDALSR (SEQ ID NO:237); if S2 comprises GTC, F2 comprises the amino acid sequence DRSALAR (SEQ ID NO: 184); and selecting the F3 zinc finger such that it binds to the S3 target subsite, wherein if S3 comprises GAA, F3 comprises the amino acid sequence QSGNLAR (SEQ ID NO:801); if S3 comprises GAG, F3 comprises the amino acid sequence RSDNLTR (SEQ ID NO:231); if S3 comprises GAC, F3 comprises the amino acid sequence DRSNLTR (SEQ ID NO:395); if S3 comprises GAT, F3 comprises the amino acid sequence TSANLSR (SEQ ID NO:377); if S3 comprises GGA, F3 comprises the amino acid sequence QSGHLQR (SEQ ID NO:287); if S3 comprises GGG, F3 comprises RSDHLSR (SEQ ID NO:229); if S3 comprises GGT, F3 comprises the amino acid sequence TSGHLVR (SEQ ID NO:1425); if S3 comprises GCA, F3 comprises the amino acid sequence QSGDLTR (SEQ ID NO:220); if S3 comprises GCG, F3 comprises the amino acid sequence RSDDLTR (SEQ ID NO:188); if S3 comprises GCC, F3 comprises the amino acid sequence DRSDLTR (SEQ ID NO:417); if S3 comprises GCT, F3 comprises the amino acid sequence QSSDLQR (SEQ ID NO:132); if S3 comprises GTG, F3 comprises RSDALTR (SEQ ID NO:153); and if S3 comprises GTC, F3 comprises the amino acid sequence DRSALAR (SEQ ID NO:184);

wherein the introduced polynucleotide expresses the zinc finger protein, and zinc finger protein binds to the target site, thereby modulating expression of the gene.

2. The method of claim 1, wherein the zinc finger protein further comprises a regulatory domain.