DNA-BINDING PROTEIN USING PPR MOTIF, AND USE THEREOF

Abstract

The object of the present invention is to, by analyzing PPR proteins that act to bind to DNA with a prediction that RNA recognition rules of PPR motifs can also be used for recognition of DNA, find a PPR protein showing such a characteristic. According to the present invention, it was revealed that, with a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, which contains one or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15, of PPR motifs having a structure of the following formula 1 (wherein, in the formula 1, Helix A is a part that can form an .alpha.-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an .alpha.-helix structure; and L is a part consisting of 2 to 7 amino acids), and having a specific combination of amino acids corresponding to a DNA base or DNA base sequence as amino acids of three positions of No. 1 A.A., No. 4 A.A., in Helix A of the formula 1 and No. "ii" (-2) A.A. contained in L of the formula 1, the aforementioned object could be achieved. (Helix A)-X-(Helix B)-L (Formula 1)

Description

CROSS-REFERENCE OF RELATED APPLICATIONS

[0001] This application is a Divisional of application Ser. No. 16/216,617, filed Dec. 11, 2018, which is a Divisional of application Ser. No. 14/785,952, filed Oct. 21, 2015, which is a 371 of International Application No. PCT/JP2014/061329, filed Apr. 22, 2014, which claims priority of Japanese Patent Application No. 2013-089840, filed Apr. 22, 2013, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to a protein that can selectively or specifically bind to an intended DNA base or DNA sequence. According to the present invention, a pentatricopeptide repeat (PPR) motif is utilized. The present invention can be used for identification and design of a DNA-binding protein, identification of a target DNA of a protein having a PPR motif, and functional control of DNA. The present invention is useful in the fields of medicine, agricultural science, and so forth. The present invention also relates to a novel DNA-cleaving enzyme that utilizes a complex of a protein containing a PPR motif and a protein that defines a functional region.

BACKGROUND ART

[0003] In recent years, techniques of binding nucleic acid-binding protein factors elucidated through various analyses to an intended sequence have been established, and they are coming to be used. Use of this sequence-specific binding is enabling analysis of intracellular localization of a target nucleic acid (DNA or RNA), elimination of a target DNA sequence, or expression control (activation or inactivation) of a protein-encoding gene existing downstream of a target DNA sequence. There are being conducted researches and developments using the zinc finger protein (Non-patent documents 1 and 2), TAL effecter (TALE, Non-patent document 3, Patent document 1), and CRISPR (Non-patent documents 4 and 5) as protein factors that act on DNA as materials for protein engineering. However, types of such protein factors are still extremely limited.

[0004] For example, the artificial enzyme, zinc finger nuclease (ZFN), known as an artificial DNA-cleaving enzyme, is a chimera protein obtained by binding a part that is constituted by linking 3 to 6 zinc fingers that specifically recognize a DNA consisting of 3 or 4 nucleotides and bind to it, and recognizes a nucleotide sequence in a sequence unit of 3 or 4 nucleotides with one DNA cleavage domain of a bacterial DNA-cleaving enzyme (for example, FokI) (Non-patent document 2). In such a chimera protein, the zinc finger domain is a protein domain that is known to bind to DNA, and it is based on the knowledge that many transcription factors have the aforementioned domain, and bind to a specific DNA sequence to control expression of a gene. By using two of ZFNs each having three zinc fingers, cleavage of one site per 70 billion nucleotides can be induced in theory.

[0005] However, because of the high cost required for the production of ZFNs, etc., the methods using ZFNs have not come to be widely used yet. Moreover, functional sorting efficiency of ZFNs is bad, and it is suggested that the methods have a problem also in this respect. Furthermore, since a zinc finger domain consisting of n of zinc fingers tends to recognize a sequence of (GNN)n, the methods also have a problem that degree of freedom for the target gene sequence is low.

[0006] An artificial enzyme, TALEN, has also been developed by binding a protein consisting of a combinatory sequence of module parts that can recognize every one nucleotide, TAL effecter (TALE), with a DNA cleavage domain of a bacterial DNA-cleaving enzyme (for example, FokI), and it is being investigated as an artificial enzyme that can replace ZFNs (Non-patent document 3). This TALEN is an enzyme generated by fusing a DNA binding domain of a transcription factor of a plant pathogenic Xanthomonas bacterium, and the DNA cleavage domain of the DNA restriction enzyme FokI, and it is known to bind to a neighboring DNA sequence to form a dimer and cleave a double strand DNA. Since, as for this molecule, the DNA binding domain of TALE found from a bacterium that infects with plants recognize one base with a combination of amino acids at two sites in the TALE motif consisting of 34 amino acid residues, it has a characteristic that binding property for a target DNA can be chosen by choosing the repetitive structure of the TALE module. TALEN using the DNA binding domain that has such a characteristic as mentioned above has a characteristic that it enables introduction of mutation into a target gene, like ZFNs, but the significant superiority thereof to ZFNs is that degree of freedom for the target gene (nucleotide sequence) is markedly improved, and the nucleotide to which it binds can be defined with a code.

[0007] However, since the total conformation of TALEN has not been elucidated, the DNA cleavage site of TALEN has not been identified at present. Therefore, it has a problem that cleavage site of TALEN is inaccurate, and is not fixed, compared with ZFNs, and it also cleaves even a similar sequence. Therefore, it has a problem that a nucleotide sequence cannot be accurately cleaved at an intended target site with a DNA-cleaving enzyme. For these reasons, it is desired to develop and provide a novel artificial DNA-cleaving enzyme free from the aforementioned problems.

[0008] On the basis of genome sequence information, PPR proteins (proteins having a pentatricopeptide repeat (PPR) motif) constituting a big family of no less than 500 members only for plants have been identified (Non-patent document 6). The PPR proteins are nucleus-encoded proteins, but are known to act on or involved in control, cleavage, translation, splicing, RNA edition, and RNA stability chiefly at an RNA level in organelles (chloroplasts and mitochondria) in a gene-specific manner. The PPR proteins typically have a structure consisting of about 10 contiguous 35-amino acid motifs of low conservativeness, i.e., PPR motifs, and it is considered that the combination of the PPR motifs is responsible for the sequence-selective binding with RNA. Almost all the PPR proteins consist only of repetition of about 10 PPR motifs, and any domain required for exhibiting a catalytic action is not found in many cases. Therefore, it is considered that the PPR proteins are essentially RNA adapters (Non-patent document 7).

[0009] In general, binding of a protein and DNA, and binding of a protein and RNA are attained by different molecular mechanisms. Therefore, a DNA-binding protein generally does not bind to RNA, whereas an RNA-binding protein generally does not bind to DNA. For example, in the case of the pumilio protein, which is known as an RNA-binding factor, and can encode RNA to be recognized, binding thereof to DNA has not been reported (Non-patent documents 8 and 9).

[0010] However, in the process of investigating properties of various kinds of PPR proteins, it became clear that it could be suggested that some types of the PPR proteins worked as DNA-binding factors.

[0011] The wheat p63 is a PPR protein having 9 PPR motifs, and it is suggested by gel shift assay that it binds to DNA in a sequence-specific manner (Non-patent document 10).

[0012] The GUN1 protein of Arabidopsis thaliana has 11 PPR motifs, and it is suggested by pull down assay that it binds with DNA (Non-patent document 11).

[0013] It has been demonstrated by run-on assay that the Arabidopsis thaliana pTac2 (protein having 15 PPR motifs, Non-patent document 12) and Arabidopsis thaliana DG1 (protein having 10 PPR motifs, Non-patent document 12) directly participate in transcription for generating RNA by using DNA as a template, and they are considered to bind to DNA.

[0014] An Arabidopsis thaliana strain deficient in the gene of GRP23 (protein having 11 PPR motifs, Non-patent document 14) shows the phenotype of embryonal death. It has been demonstrated that this protein physically interacts with the major subunit of the eukaryotic RNA transcription polymerase 2, which is a DNA-dependent RNA transcription enzyme, and therefore it is considered that GRP23 also acts to bind to DNA.

[0015] However, bindings of these PPR proteins to DNA have been only indirectly suggested, and actual sequence-specific binding has not been fully verified. Moreover, even if such proteins bind with DNA, it is generally considered that binding of a protein and DNA, and binding of a protein and RNA are attained by different molecular mechanisms, and therefore what kind of sequence rule specifically exists, with which binding is attained, etc., are not even expected at all.

PRIOR ART REFERENCES

Patent Documents

[0016] Patent document 1: WO2011/072246

[0017] Patent document 2: WO2011/111829

Non-Patent Documents

[0017]

[0018] Non-patent document 1: Maeder, M. L., et al. (2008) Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification, Mol. Cell 31, 294-301

[0019] Non-patent document 2: Urnov, F. D., et al. (2010) Genome editing with engineered zinc finger nucleases, Nature Review Genetics, 11, 636-646

[0020] Non-patent document 3: Miller, J. C., et al. (2011) A TALE nuclease architecture for efficient genome editing, Nature Biotech., 29, 143-148

[0021] Non-patent document 4: Mali P., et al. (2013) RNA-guided human genome engineering via Cas9, Science, 339, 823-826

[0022] Non-patent document 5: Cong L., et al. (2013) Multiplex genome engineering using CRISPR/Cas systems, Science, 339, 819-823

[0023] Non-patent document 6: Small, I. D. and Peeters, N. (2000) The PPR motif --a TPR-related motif prevalent in plant organellar proteins, Trends Biochem. Sci., 25, 46-47

[0024] Non-patent document 7: Woodson, J. D., and Chory, J. (2008) Coordination of gene expression between organellar and nuclear genomes, Nature Rev. Genet., 9, 383-395

[0025] Non-patent document 8: Wang, X., et al. (2002) Modular recognition of RNA by a human pumilio-homology domain, Cell, 110, 501-512

[0026] Non-patent document 9: Cheong, C. G, and Hall and T. M. (2006) Engineering RNA sequence specificity of Pumilio repeats, Proc. Natl. Acad. Sci. USA 103, 13635-13639

[0027] Non-patent document 10: Ikeda T. M. and Gray M. W. (1999) Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria, Mol. Cell Bio., 119 (12): 8113-8122

[0028] Non-patent document 11: Koussevitzky S., et al. (2007) Signals from chloroplasts converge to regulate nuclear gene expression, Science, 316:715-719

[0029] Non-patent Document 12: Pfalz J, et al. (2006) PTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression, Plant Cell 18:176-197

[0030] Non-patent document 13: Chi W, et al. (2008) The pentatricopeptide repeat protein DELAYED GREENING1 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis, Plant Physiol., 147:573-584

[0031] Non-patent document 14: Ding Y H, et al. (2006) Arabidopsis GLUTAMINE-RICH PROTEIN 23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III, Plant Cell, 18:815-830

SUMMARY OF THE INVENTION

Object to be Achieved by the Invention

[0032] The inventors of the present invention expected that the properties of the PPR proteins (proteins having a PPR motif) as RNA adapters would be determined by property of each PPR motif constituting the PPR proteins and combination of a plurality of PPR motifs, and proposed methods for modifying RNA-binding proteins using such PPR motifs (Patent document 2). Then, they elucidated that a PPR motif and RNA bind in one-to-one correspondence, contiguous PPR motifs recognize contiguous RNA bases in an RNA sequence, and such RNA recognition is determined by combination of amino acids at specific three positions among the 35 amino acids constituting the PPR motif, and filed a patent application for a method for designing a customized RNA-binding protein utilizing RNA recognition codes of PPR motifs and use thereof (PCT/JP2012/077274; Yagi, Y., et al. (2013) PLoS One, 8, e57286; and Barkan, A., et al. (2012) PLoS Genet., 8, e1002910).

[0033] It has been generally considered that binding of a protein and DNA, and binding of a protein and RNA are attained by different molecular mechanisms. However, the inventors of the present invention predicted that the RNA recognition rule of the PPR motif would be also usable for recognition of DNA, and analyzed PPR proteins that act to bind with DNA aiming at retrieving PPR proteins having such a characteristic. They also aimed at providing a novel artificial enzyme by preparing a customized DNA-binding protein that binds to a desired sequence using such a PPR protein that specifically binds to a DNA obtained as described above, and using it with a protein that defines a functional region, and providing a novel artificial DNA-cleaving enzyme by using it together with a region having a DNA-cleaving activity as the functional region.

Means for Achieving the Object

[0034] As for the PPR proteins, it was elucidated by various domain search programs (Pfam, Prosite, Interpro, etc.) that the PPR motifs contained in the common RNA-binding type PPR proteins and the PPR motifs contained in the DNA-binding PPR proteins of some kinds mentioned above are not particularly distinguished. Therefore, it was considered that PPR proteins might contain amino acids (amino acid group) that would determine a binding property for DNA or a binding property for RNA apart from the amino acids required for the nucleic acid recognition.

[0035] The inventors of the present invention elucidated that an RNA-binding PPR motif and RNA bind in one-to-one correspondence, contiguous PPR motifs recognize contiguous RNA bases in an RNA sequence, and in such recognition, base-selective binding with RNA is determined by combination of RNA recognition amino acids at specific three positions (that is, the first and fourth amino acids of the first helix (Helix A) among the two .alpha.-helix structures constituting the motif (No. 1 A.A. and No. 4 A.A.), and the second amino acid counted from the C-terminus (No. "ii" (-2) A.A.)), among the 35 amino acids constituting the PPR motif, and filed a patent application for a method for designing a customized RNA-binding protein utilizing RNA recognition codes of PPR motifs and use thereof (PCT/JP2012/077274).

[0036] Then, among the PPR proteins, for the aforementioned wheat p63 (Non-patent document 11, the amino acid sequence of the homologous protein of Arabidopsis thaliana is shown as SEQ ID NO: 1), GUN1 protein of Arabidopsis thaliana (Non-patent document 12, amino acid sequence thereof is shown as SEQ ID NO: 2), pTac2 of Arabidopsis thaliana (Non-patent document 13, amino acid sequence thereof is shown as SEQ ID NO: 3), DG1 (Non-patent document 14, amino acid sequence thereof is shown as SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana (Non-patent document 15, amino acid sequence thereof is shown as SEQ ID NO: 5), for which binding with DNA was suggested, amino acid frequencies of the amino acids at three positions bearing the nucleic acid recognition codes in the PPR motif considered to be important when RNA is a target (No. 1 A.A., No. 4 A.A. and No. "ii" (-2) A.A.) were compared with those found in the RNA binding type motif. As a result, it became clear that the tendencies of the amino acid frequencies found in those PPR motifs as mentioned above, for which DNA-binding property was suggested, and the RNA binding type motifs substantially agreed with each other.

[0037] The above results suggest that the nucleic acid recognition codes of the RNA binding type PPR motifs can also be applied to the DNA binding type PPR motifs. Thymine (T) is a uracil (U) derivative having a structure consisting of uracil (U) of which carbon of the 5-position is methylated, as it is also called 5-methyluracil. Such a characteristic of the base constituting the nucleic acid suggests that the combination of the amino acids that recognizes uracil (U) of an RNA binding type PPR motif is used for recognition of thymine (T) in DNA.

[0038] On the basis of the aforementioned findings, it was elucidated that, by using the aforementioned p63 (amino acid sequence of SEQ ID NO: 1), GUN1 protein of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 2), pTac2 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 3), DG1 (amino acid sequence of SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 5), which are DNA-binding type PPR proteins, as a template, arranging amino acids of the three positions (No. 1 A.A., No. 4 A.A. and No. "ii" (-2) A.A.) with applying the finding obtained for such PPR proteins as a result of examination of the RNA-binding type PPR motifs, a customized DNA-binding protein that binds to an arbitrary DNA base sequence could be produced.

[0039] That is, the inventors of the present invention provided a protein that comprises 2 or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15, of PPR motifs having the specific amino acids described later as the amino acids at the three positions (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) in the PPR motifs, and can bind to DNA in a DNA base-selective manner or DNA base sequence-selective manner, of which typical examples are the amino acid sequences of SEQ ID NOS: 1 to 5, and thus accomplished the present invention.

[0040] The present invention provides the followings.

[0041] [1] A method for modifying a genetic substance of a cell, the method including: designing a DNA binding protein; determining a DNA base sequence coding for an amino acid sequence of a fused protein comprising a functional region and a DNA binding region consisting of a DNA-binding protein; cloning said DNA base sequence; preparing a vector carrying said DNA base sequence and a cell containing a DNA having at least 9 target DNA bases as a target DNA base sequence; and introducing the vector into the cell so that the DNA binding region of the fused protein binds to the DNA having the at least 9 target DNA bases, and therefore the functional region modifies the DNA having the at least 9 target DNA bases, wherein the DNA-binding protein contains at least 9 PPR motifs for binding to the at least 9 target DNA bases, respectively, and having a structure of the following formula 1: [Formula 1]

(Helix A)-X-(Helix B)-L (Formula 1)

(wherein, in the formula 1: Helix A is a part that can form an .alpha.-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an .alpha.-helix structure; and L is a part consisting of 2 to 7 amino acids), wherein, position numbers of amino acids in the PPR motifs are the same as PF01535 in Pfam under the following definitions: the first amino acid of Helix A is referred to as No. 1 amino acid (No. 1 A.A.), the fourth amino acid as No. 4 amino acid (No. 4 A.A.), and

[0042] when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (when there is no amino acid insertion between the PPR motifs), the -2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n);

[0043] when a non-PPR motif consisting of 1 to 20 amino acids exists between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the -2nd amino acid; or

[0044] when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n) is referred to as No. "ii" (-2) amino acid (No. "ii" (-2) A.A.), one PPR motif (M.sub.n) contained in the protein is a PPR motif having a specific combination of amino acids corresponding to a target DNA base or target DNA base sequence as the three amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.

[2] The protein according to [1], wherein the combination of the three amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. is a combination corresponding to a target DNA base or target DNA base sequence. In one embodiment, each PPR motif (M.sub.n) contained in the DNA-binding protein is a PPR motif having a specific combination of amino acids as the three amino acids of Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, such that the DNA-binding protein has at least 9 combinations of the three amino acids corresponding to the respective target DNA bases of the target DNA base sequence. Each combination of the three amino acids is determined according to any one of the following definitions: (1-1) when No. 4 A.A. is glycine (G), No. 1 A.A. may be an arbitrary amino acid, and No. "ii" (-2) A.A. is aspartic acid (D), asparagine (N), or serine (S); (1-2) when No. 4 A.A. is isoleucine (I), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-3) when No. 4 A.A. is leucine (L), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-4) when No. 4 A.A. is methionine (M), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-5) when No. 4 A.A. is asparagine (N), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-6) when No. 4 A.A. is proline (P), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-7) when No. 4 A.A. is serine (S), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; (1-8) when No. 4 A.A. is threonine (T), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid; and (1-9) when No. 4 A.A. is valine (V), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid. [3] The protein according to [1], wherein the combination of the three amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. is a combination corresponding to a target DNA base or target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions: (2-1) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are an arbitrary amino acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-2) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-3) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; (2-4) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glutamic acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; (2-5) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, glycine, and serine, respectively, the PPR motif selectively binds to A, and next binds to C; (2-6) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, isoleucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; (2-7) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, isoleucine, and asparagine, respectively, the PPR motif selectively binds to T, and next binds to C; (2-8) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; (2-9) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and aspartic acid, respectively, the PPR motif selectively binds to C; (2-10) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and lysine, respectively, the PPR motif selectively binds to T; (2-11) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, methionine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; (2-12) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-13) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-14) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to C and T; (2-15) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-16) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-17) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glycine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-18) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-19) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are threonine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-20) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are valine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-21) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are tyrosine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-22) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-23) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-24) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are serine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-25) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-26) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and serine, respectively, the PPR motif selectively binds to C; (2-27) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and serine, respectively, the PPR motif selectively binds to C; (2-28) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and threonine, respectively, the PPR motif selectively binds to C; (2-29) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and threonine, respectively, the PPR motif selectively binds to C; (2-30) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and tryptophan, respectively, the PPR motif selectively binds to C, and next binds to T; (2-31) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and tryptophan, respectively, the PPR motif selectively binds to T, and next binds to C; (2-32) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, proline, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; (2-33) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-34) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-35) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are tyrosine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-36) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, serine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; (2-37) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-38) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-39) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-40) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; (2-41) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-42) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-43) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-44) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-45) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-46) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-47) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and an arbitrary amino acid, respectively, the PPR motif binds with A, C, and T, but does not bind to G; (2-48) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, valine, and aspartic acid, respectively, the PPR motif selectively binds to C, and next binds to A; (2-49) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and glycine, respectively, the PPR motif selectively binds to C; and (2-50) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and threonine, respectively, the PPR motif selectively binds to T. [4] The protein according to any one of [1] to [3], which contains 2 to 30 of the PPR motifs (M.sub.n) defined in [1]. [5] The protein according to any one of [1] to [3], which contains 5 to 25 of the PPR motifs (M.sub.n) defined in [1]. [6] The protein according to any one of [1] to [3], which contains 9 to 15 of the PPR motifs (M.sub.n) defined in [1]. [7] The PPR protein according to [6], which consists of a sequence selected from the amino acid sequence of SEQ ID NO: 1 containing 9 PPR motifs, the amino acid sequence of SEQ ID NO: 2 containing 11 PPR motifs, the amino acid sequence of SEQ ID NO: 3 containing 15 PPR motifs, the amino acid sequence of SEQ ID NO: 4 containing 10 PPR motifs, and the amino acid sequence of SEQ ID NO: 5 containing 11 PPR motifs. [8] A method for identifying a DNA base or DNA base sequence that serves as a target of a DNA-binding protein containing one or more (preferably 2 to 30) PPR motifs (M.sub.n) defined in [1], wherein:

[0045] the DNA base or DNA base sequence is identified by determining presence or absence of a DNA base corresponding to a combination of the three amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. of the PPR motif on the basis of any one of the definitions (1-1) to (1-9) mentioned in [2], and (2-1) to (2-50) mentioned in [3]. [9] A method for identifying a PPR protein containing one or more (preferably 2 to 30) PPR motifs (M.sub.n) defined in [1] that can bind to a target DNA base or target DNA having a specific base sequence, wherein:

[0046] the PPR protein is identified by determining presence or absence of a combination of the three amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. corresponding to the target DNA base or a specific base constituting the target DNA on the basis of any one of the definitions (1-1) to (1-9) mentioned in [2], and (2-1) to (2-50) mentioned in [3].

[10] A method for controlling a function of DNA, which uses the protein according to [1]. [11] A complex consisting of a region comprising the protein according to [1], and a functional region bound together. [12] The complex according to [11], wherein the functional region is fused to the protein according to [1] on the C-terminus side of the protein. [13] The complex according to [11] or [12], wherein the functional region is a DNA-cleaving enzyme, or a nuclease domain thereof, or a transcription control domain, and the complex functions as a target sequence-specific DNA-cleaving enzyme or transcription control factor. [14] The complex according to [13], wherein the DNA-cleaving enzyme is the nuclease domain of FokI (SEQ ID NO: 6). [15] A method for modifying a genetic substance of a cell comprising the following steps:

[0047] preparing a cell containing a DNA having a target sequence; and

[0048] introducing the complex according to [11] into the cell so that the region of the complex consisting of the protein binds to the DNA having a target sequence, and therefore the functional region modifies the DNA having a target sequence.

[16] A method for identifying, recognizing, or targeting a DNA base or DNA having a specific base sequence by using a PPR protein containing one or more PPR motifs. [17] The method according to [16], wherein the protein contains one or more PPR motifs in which three amino acids among the amino acids constituting the motif constitute a specific combination of amino acids. [18] The method according to [16] or [17], wherein the protein contains one or more PPR motifs (M.sub.n) defined in [1].

Effect of the Invention

[0049] According to the present invention, a PPR motif that can binds to a target DNA base, and a protein containing it can be provided. By arranging two or more PPR motifs, a protein that can binds to a target DNA having an arbitrary sequence or length can be provided.

[0050] According to the present invention, a target DNA of an arbitrary PPR protein can be predicted and identified, and conversely, a PPR protein that binds to an arbitrary DNA can be predicted and identified. Prediction of such a target DNA sequence clarifies the genetic identity thereof, and increases possibility of use thereof. Furthermore, according to the present invention, functionalities of homologous genes of a gene of an industrially useful PPR protein showing amino acid polymorphism at a high level can be determined on the basis of difference of the target DNA base sequences thereof.

[0051] Furthermore, according to the present invention, a novel DNA-cleaving enzyme using a PPR motif can also be provided. That is, by linking a protein as a functional region with the PPR motif or PPR protein provided by the present invention, a complex containing a protein having a binding activity for a specific nucleic acid sequence, and a protein having a specific functionality can be prepared.

[0052] The functional region usable in the present invention is one that can impart, among various functions, a function for any one of cleavage, transcription, replication, restoration, synthesis, modification, etc. of DNA. By choosing the sequence of the PPR motifs, which is the characteristic of the present invention, to determine a base sequence of DNA as a target, almost all DNA sequences can be used as a target, and genome edition using a function of the functional region such as those for cleavage, transcription, replication, restoration, synthesis, modification, etc. of DNA can be realized with such a target.

[0053] For example, when the functional region has a function for cleaving DNA, a complex comprising a PPR protein part prepared according to the present invention and a DNA-cleaving region linked together is provided. Such a complex can function as an artificial DNA-cleaving enzyme, which recognizes a base sequence of DNA as a target with the PPR protein part, and then cleaves DNA with the region for cleaving DNA. When the functional region has a transcription control function, a complex comprising a PPR protein part prepared according to the present invention and a transcription control region for DNA linked together is provided. Such a complex can function as an artificial transcription control factor, which recognizes a base sequence of DNA as a target with the PPR protein part, and then promotes transcription of the target DNA.

[0054] The present invention can further be utilized for a method for delivering the aforementioned complex in a living body so that the complex functions in the living body, and preparation of transformants utilizing a nucleic acid sequence (DNA and RNA) encoding a protein obtained according to the present invention, as well as specific modification, control, and impartation of a function in various situations in organisms (cells, tissues, and individuals).

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIGS. 1A-1C show conserved sequences and amino acid numbers of the PPR motif. FIG. 1A shows the amino acids constituting the PPR motif defined in the present invention, and the amino acid numbers thereof (the amino acid sequences P, S, L1, and L2 correspond to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively). FIG. 1B shows positions of three amino acids (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) that control binding base selectivity in the predicted structure. FIG. 1C shows two examples of the structure of the PPR motif, and the positions of the amino acids on the predicted structure for each case. No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are indicated with sticks of magenta color (dark gray in the case of monochratic display) in the conformational diagrams of the protein.

[0056] FIG. 2 summarizes the outlines of the structures of Arabidopsis thaliana p63 (amino acid sequence of SEQ ID NO: 1), the GUN1 protein of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 2), pTac2 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 3), DG1 (amino acid sequences of SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 5), which are DNA-binding type PPR proteins that function in DNA metabolism, and the outline of the assay system for demonstrating that they bind to DNA.

[0057] FIG. 3 summarizes the amino acid frequencies of the amino acids at the three positions bearing the nucleic acid recognition codes in the PPR motif (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) for the PPR motifs of the PPR proteins (SEQ ID NOS: 1 to 5), for which DNA binding property was suggested, and known RNA-binding type motifs.

[0058] FIG. 4-1 shows the positions of the PPR motifs included in the inside of the proteins, and the positions of the three amino acids bearing the nucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) in the PPR motifs for each of (A) Arabidopsis thaliana p63 (amino acid sequence of SEQ ID NO: 1) and (B) the GUN1 protein of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 2.

[0059] FIG. 4-2 shows the positions of the PPR motifs included in the inside of the proteins, and the positions of the three amino acids bearing the nucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) in the PPR motifs for each of (C) pTac2 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 3), and (D) DG1 (amino acid sequence of SEQ ID NO: 4).

[0060] FIG. 4-3 shows the positions of the PPR motifs included in the inside of the proteins, and the positions of the three amino acids bearing the nucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) in the PPR motifs for (E) GRP23 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 5).

[0061] FIG. 5 shows the evaluation of the sequence-specific DNA-binding abilities of the PPR molecules. Artificial transcription factors were prepared by fusing each of three kinds of DNA-binding type (regarded so) PPR molecules with VP64, which is a transcription activation domain, and whether they could activate a luciferase reporter having each target sequence was examined in a human cultured cell.

[0062] FIG. 6 shows comparison of the luciferase activities observed by cointroduction of pTac2-VP64 or GUN1-VP64 with pminCMV-luc2 as a negative control, or a reporter vector comprising 4 or 8 target sequences. As a result, there was observed a tendency that the activity increased with increase of the target sequence for the both molecules, and thus it was verified that these PPR-VP64 molecules specifically bound to each target sequence to function as a site-specific transcription activator.

MODES FOR CARRYING OUT THE INVENTION

[0063] [PPR Motif and PPR Protein]

[0064] The "PPR motif" referred to in the present invention means a polypeptide constituted with 30 to 38 amino acids and having an amino acid sequence that shows, when the amino acid sequence is analyzed with a protein domain search program on the web (for example, Pfam, Prosite, Uniprot, etc.), an E value not larger than a predetermined value (desirably E-03) obtained at PF01535 in the case of Pfam (http://pfam.sanger.ac.uk/), or PS51375 in the case of Prosite (http://www.expasy.org/prosite/), unless otherwise indicated. The PPR motifs in various proteins are also defined in the Uniprot database (http://www.uniprot.org).

[0065] Although the amino acid sequence of the PPR motif is not highly conserved in the PPR motif of the present invention, such a secondary structure of helix, loop, helix, and loop as shown by the following formula is conserved well.

[Formula 2]

(Helix A)-X-(Helix B)-L (Formula 1)

[0066] The position numbers of the amino acids constituting the PPR motif defined in the present invention are according to those defined in a paper of the inventors of the present invention (Kobayashi K, et al., Nucleic Acids Res., 40, 2712-2723 (2012)). That is, the position numbers of the amino acids constituting the PPR motif defined in the present invention are substantially the same as the amino acid numbers defined for PF01535 in Pfam, but correspond to numbers obtained by subtracting 2 from the amino acid numbers defined for PS51375 in Prosite (for example, position 1 according to the present invention is position 3 of PS51375), and also correspond to numbers obtained by subtracting 2 from the amino acid numbers of the PPR motif defined in Uniprot.

[0067] More precisely, in the present invention, the No. 1 amino acid is the first amino acid from which Helix A shown in the formula 1 starts. The No. 4 amino acid is the fourth amino acid counted from the No. 1 amino acid. As for "ii" (-2)nd amino acid,

[0068] when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (when there is no amino acid insertion between the PPR motifs, as in the cases of, for example, Motif Nos. 1, 2, 3, 4, 6 and 7 in FIG. 4-1 (A)), the -2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n) is referred to as No. "ii" (-2) amino acid;

[0069] when a non-PPR motif (part that is not the PPR motif) consisting of 1 to 20 amino acids exists between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side (as in the cases of, for example, Motif Nos. 5 and 8 in FIG. 4-1 (A), and Motif Nos. 1, 2, 7 and 8 in FIG. 4-3 (D)), the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the -2nd amino acid, is referred to as No. "ii" (-2) amino acid (refer to FIG. 1); or

[0070] when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n) (as in the cases of, for example, Motif No. 9 in FIG. 4-1 (A), and Motif No. 11 in FIG. 4-1 (B)), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n) is referred to as No. "ii" (-2) amino acid.

[0071] The "PPR protein" referred to in the present invention means a PPR protein having two or more of the aforementioned PPR motifs, unless otherwise indicated. The term "protein" used in this specification means any substance consisting of a polypeptide (chain consisting of two or more amino acids bound through peptide bonds), and also includes those consisting of a comparatively low molecular weight polypeptide, unless otherwise indicated. The "amino acid" referred to in the present invention means a usual amino acid molecule, as well as an amino acid residue constituting a peptide chain. Which the term means will be apparent to those skilled in the art from the context.

[0072] Many PPR proteins exist in plants, and 500 proteins and about 5000 motifs can be found in Arabidopsis thaliana. PPR motifs and PPR proteins of various amino acid sequences also exist in many land plants such as rice, poplar, and selaginella. It is known that some PPR proteins are important factors for obtaining F1 seeds for hybrid vigor as fertility restoration factors that are involved in formation of pollen (male gamete). It has been clarified that some PPR proteins are involved in speciation, similarly in fertility restoration. It has also been clarified that almost all the PPR proteins act on RNA in mitochondria or chloroplasts.

[0073] It is known that, in animals, anomaly of the PPR protein identified as LRPPRC causes Leigh syndrom French Canadian (LSFC, Leigh's syndrome, subacute necrotizing encephalomyelopathy).

[0074] The term "selective" used for a property of a PPR motif for binding with a DNA base in the present invention means that a binding activity for any one base among the DNA bases is higher than binding activities for the other bases, unless otherwise indicates. Those skilled in the art can confirm this selectivity by planning an experiment, or it can also be obtained by calculation as described in the examples mentioned in this specification.

[0075] The DNA base referred to in the present invention means a base of deoxyribonucleotide constituting DNA, and specifically, it means any of adenine (A), guanine (G), cytosine (C), and thymine (T), unless otherwise indicated. Although the PPR protein may have selectivity to a base in DNA, it does not bind to a nucleic acid monomer.

[0076] Although search methods for conserved amino acid sequence as the PPR motif had been established before the present invention was accomplished, any rule concerning selective binding with DNA base had not been discovered at all.

Findings Provided by the Present Invention

[0077] The following findings are provided by the present invention.

(I) Information about Positions of Amino Acids Important for Selective Binding

[0078] Specifically, under the following definitions:

the first amino acid of Helix A of the PPR motif is referred to as No. 1 amino acid (No. 1 A.A.), the fourth amino acid as No. 4 amino acid (No. 4 A.A.), and

[0079] when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (when there is no amino acid insertion between the PPR motifs), the -2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n);

[0080] when a non-PPR motif consisting of 1 to 20 amino acids exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the -2nd amino acid; or

[0081] when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n)

is referred to as No. "ii" (-2) amino acid (No. "ii" (-2) A.A.), combination of the three amino acids, the first and fourth amino acids of the helix (Helix A), No. 1 and No. 4 amino acids, and No. "ii" (-2) A.A. defined above (No. 1 A.A., No. 4 A.A. and No. "ii" (-2) A.A.) is important for selective binding to a DNA base, and to what kind of DNA base the motif binds can be determined on the basis of the combination.

[0082] The present invention is based on the findings concerning the combination of the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., found by the inventors of the present invention. Specifically:

(1-1) when No. 4 A.A. is glycine (G), No. 1 A.A. may be an arbitrary amino acid, No. "ii" (-2) A.A. is aspartic acid (D), asparagine (N), or serine (S), and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0083] a combination of an arbitrary amino acid and aspartic acid (D) (*GD),

[0084] preferably a combination of glutamic acid (E) and aspartic acid (D) (EGD),

[0085] a combination of an arbitrary amino acid and asparagine (N) (*GN),

[0086] preferably a combination of glutamic acid (E) and asparagine (N) (EGN), or

[0087] a combination of an arbitrary amino acid and serine (S) (*GS);

(1-2) when No. 4 A.A. is isoleucine (I), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0088] a combination of an arbitrary amino acid and asparagine (N) (*IN);

(1-3) when No. 4 A.A. is leucine (L), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0089] a combination of an arbitrary amino acid and aspartic acid (D) (*LD), or

[0090] a combination of an arbitrary amino acid and lysine (K) (*LK);

(1-4) when No. 4 A.A. is methionine (M), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0091] a combination of an arbitrary amino acid and aspartic acid (D) (*MD), or

[0092] a combination of isoleucine (I) and aspartic acid (D) (IMD);

(1-5) when No. 4 A.A. is asparagine (N), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0093] a combination of an arbitrary amino acid and aspartic acid (D) (*ND),

[0094] a combination of any one of phenylalanine (F), glycine (G), isoleucine (I), threonine (T), valine (V) and tyrosines (Y), and aspartic acid (D) (FND, GND, IND, TND, VND, or YND),

[0095] a combination of an arbitrary amino acid and asparagine (N) (*NN),

[0096] a combination of any one of isoleucine (I), serine (S) and valine (V), and asparagine (N) (INN, SNN or VNN)

[0097] a combination of an arbitrary amino acid and serine (S) (*NS),

[0098] a combination of valine (V) and serine (S) (VNS),

[0099] a combination of an arbitrary amino acid and threonine (T) (*NT),

[0100] a combination of valine (V) and threonine (T) (VNT),

[0101] a combination of an arbitrary amino acid and tryptophan (W) (*NW), or

[0102] a combination of isoleucine (I) and tryptophan (W) (INW);

(1-6) when No. 4 A.A. is proline (P), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0103] a combination of an arbitrary amino acid and aspartic acid (D) (*PD),

[0104] a combination of phenylalanine (F) and aspartic acid (D) (FPD), or

[0105] a combination of tyrosine (Y) and aspartic acid (D) (YPD);

(1-7) when No. 4 A.A. is serine (S), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0106] a combination of an arbitrary amino acid and asparagine (N) (*SN),

[0107] a combination of phenylalanine (F) and asparagine (N) (FSN), or

[0108] a combination of valine (V) and asparagine (N) (VSN);

(1-8) when No. 4 A.A. is threonine (T), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0109] a combination of an arbitrary amino acid and aspartic acid (D) (*TD),

[0110] a combination of valine (V) and aspartic acid (D) (VTD),

[0111] a combination of an arbitrary amino acid and asparagine (N) (*TN),

[0112] a combination of phenylalanine (F) and asparagine (N) (FTN),

[0113] a combination of isoleucine (I) and asparagine (N) (ITN), or

[0114] a combination of valine (V) and asparagine (N) (VTN); and

(1-9) when No. 4 A.A. is valine (V), each of No. 1 A.A. and No. "ii" (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. "ii" (-2) A.A. may be, for example:

[0115] a combination of isoleucine (I) and aspartic acid (D) (IVD),

[0116] a combination of an arbitrary amino acid and glycine (G) (*VG), or

[0117] a combination of an arbitrary amino acid and threonine (T) (*VT).

(II) Information about Correspondence of Combination of Three Amino Acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., and DNA Base

[0118] The protein is a protein determined on the basis of, specifically, the following definitions, and having a selective DNA base-binding property:

(2-1) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-2) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-3) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; (2-4) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glutamic acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; (2-5) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, glycine, and serine, respectively, the PPR motif selectively binds to A, and next binds to C; (2-6) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, isoleucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; (2-7) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, isoleucine, and asparagine, respectively, the PPR motif selectively binds to T, and next binds to C; (2-8) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; (2-9) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and aspartic acid, respectively, the PPR motif selectively binds to C; (2-10) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, leucine, and lysine, respectively, the PPR motif selectively binds to T; (2-11) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, methionine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; (2-12) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-13) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-14) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to C and T; (2-15) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-16) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-17) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are glycine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-18) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-19) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are threonine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-20) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are valine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-21) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. are tyrosine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; (2-22) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-23) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-24) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are serine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-25) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; (2-26) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and serine, respectively, the PPR motif selectively binds to C; (2-27) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and serine, respectively, the PPR motif selectively binds to C; (2-28) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and threonine, respectively, the PPR motif selectively binds to C; (2-29) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, asparagine, and threonine, respectively, the PPR motif selectively binds to C; (2-30) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, asparagine, and tryptophan, respectively, the PPR motif selectively binds to C, and next binds to T; (2-31) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, asparagine, and tryptophan, respectively, the PPR motif selectively binds to T, and next binds to C; (2-32) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, proline, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; (2-33) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-34) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-35) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are tyrosine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; (2-36) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, serine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; (2-37) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-38) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-39) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, serine, and asparagine, respectively, the PPR motif selectively binds to A; (2-40) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; (2-41) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-42) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; (2-43) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-44) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are phenylalanine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-45) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-46) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are valine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; (2-47) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and an arbitrary amino acid, respectively, the PPR motif binds with A, C, and T, but does not bind to G; (2-48) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are isoleucine, valine, and aspartic acid, respectively, the PPR motif selectively binds to C, and next binds to A; (2-49) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and glycine, respectively, the PPR motif selectively binds to C; and (2-50) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A., are an arbitrary amino acid, valine, and threonine, respectively, the PPR motif selectively binds to T.

[0119] Combination of amino acids of specific positions and binding property with a DNA base can be confirmed by experiments. Experiments for such purposes include preparation of a PPR motif or a protein containing two or more PPR motifs, preparation of a substrate DNA, and binding property test (for example, gel shift assay). These experiments are well known to those skilled in the art, and as for more specific procedures and conditions, for example, Patent document 2 can be referred to.

[0120] [Use of PPR Motif and PPR Protein]

[0121] Identification and Design

[0122] One PPR motif recognizes a specific one kind of base of DNA, and two or more contiguous PPR motifs can recognize continuous bases in a DNA sequence. Further, according to the present invention, by appropriately choosing amino acids at specific positions, PPR motifs selective for each of A, T, and C can be chosen or designed, and a protein containing an appropriate continuation of such PPR motifs can recognize a corresponding specific sequence. Therefore, according to the present invention, a naturally occurring PPR protein that selectively binds to DNA having a specific base sequence can be predicted or identified, or conversely, DNA as a target of binding of a PPR protein can be predicted and identified. Prediction or identification of such a target is useful for clarifying genetic identity of the target, and is also useful from a viewpoint that such prediction or identification may expand applicability of the target.

[0123] Furthermore, according to the present invention, a PPR motif that can selectively bind to a desired DNA base, and a protein having two or more PPR motifs that can bind to a desired DNA in a sequence-specific manner can be designed. In such design, as for the part other than the amino acids at the important positions in the PPR motif, sequence information on PPR motifs of naturally occurring type in DNA-binding type PPR proteins such as those of SEQ ID NOS: 1 to 5 can be referred to. Further, the motif or protein may also be designed by using a motif or protein of naturally occurring type as a whole, and replacing only the amino acids of the corresponding positions. Although the number of repetitions of PPR motifs can be appropriately chosen according to a target sequence, it may be, for example, 2 or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15.

[0124] In the designing, amino acids other than those of the combination of the amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. may be taken into consideration. For example, selection of the amino acids of No. 8 and No. 12 described in Patent document 2 mentioned above may be important for exhibiting a DNA-binding activity. According to the researches of the inventors of the present invention, the No. 8 amino acid of a certain PPR motif and the No. 12 amino acid of the same PPR motif may cooperate in binding with DNA. The No. 8 amino acid may be a basic amino acid, preferably lysine, or an acidic amino acid, preferably aspartic acid, and the No. 12 amino acid may be a basic amino acid, neutral amino acid, or hydrophobic amino acid.

[0125] A designed motif or protein can be prepared by methods well known to those skilled in the art. That is, the present invention provides a PPR motif that selectively binds to a specific DNA base, and a PPR protein that specifically binds to DNA having a specific sequence, in which attention is paid to the combination of the amino acids of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. Such a motif and protein can be prepared even in a comparatively large amount by methods well known to those skilled in the art, and such methods may comprise determining a nucleic acid sequence encoding a target motif or protein from the amino acid sequence of the target motif or protein, cloning it, and preparing a transformant that produces the target motif or protein.

[0126] Preparation of Complex and Use Thereof

[0127] The PPR motif or PPR protein provided by the present invention can be made into a complex by binding a functional region. The functional region generally refers to a part having such a function as a specific biological function exerted in a living body or cell, for example, enzymatic function, catalytic function, inhibitory function, promotion function, etc, or a function as a marker. Such a region consists of, for example, a protein, peptide, nucleic acid, physiologically active substance, or drug.

[0128] According to the present invention, by binding a functional region to the PPR protein, the target DNA sequence-binding function exerted by the PPR protein, and the function exerted by the functional region can be exhibited in combination. For example, if a protein having a DNA-cleaving function (for example, restriction enzyme such as FokI) or a nuclease domain thereof is used as the functional region, the complex can function as an artificial DNA-cleaving enzyme.

[0129] In order to produce such a complex, methods generally available in this technical field can be used, and there are known a method of synthesizing such a complex as one protein molecule, a method of separately synthesizing two or more members of proteins, and then combining them to form a complex, and so forth.

[0130] In the case of the method of synthesizing a complex as one protein molecule, for example, a protein complex can be designed so as to comprise a PPR protein and a cleaving enzyme bound to the C-terminus of the PPR protein via an amino acid linker, an expression vector structure for expressing the protein complex can be constructed, and the target complex can be expressed from the structure. As such a preparation method, the method described in Japanese Patent Application No. 2011-242250, and so forth can be used.

[0131] For binding the PPR protein and the functional region protein, any binding means known in this technical field may be used, including binding via an amino acid linker, binding utilizing specific affinity such as binding between avidin and biotin, binding utilizing another chemical linker, and so forth.

[0132] The functional region usable in the present invention refers to a region that can impart any one of various functions such as those for cleavage, transcription, replication, restoration, synthesis, or modification of DNA, and so forth. By choosing the sequence of the PPR motif to define a DNA base sequence as a target, which is the characteristic of the present invention, substantially any DNA sequence may be used as the target, and with such a target, genome edition utilizing the function of the functional region such as those for cleavage, transcription, replication, restoration, synthesis, or modification of DNA can be realized.

[0133] For example, when the function of the functional region is a DNA cleavage function, there is provided a complex comprising a PPR protein part prepared according to the present invention and a DNA cleavage region bound together. Such a complex can function as an artificial DNA-cleaving enzyme that recognizes a base sequence of DNA as a target by the PPR protein part, and then cleaves DNA by the DNA cleavage region.

[0134] An example of the functional region having a cleavage function usable for the present invention is a deoxyribonuclease (DNase), which functions as an endodeoxyribonuclease. As such a DNase, for example, endodeoxyribonucleases such as DNase A (e.g., bovine pancreatic ribonuclease A, PDB 2AAS), DNase H and DNase I, restriction enzymes derived from various bacteria (for example, FokI (SEQ ID NO: 6) etc.) and nuclease domains thereof can be used. Such a complex comprising a PPR protein and a functional region does not exist in the nature, and is novel.

[0135] When the function of the functional region is a transcription control function, there is provided a complex comprising a PPR protein part prepared according to the present invention and a DNA transcription control region bound together. Such a complex can function as an artificial transcription control factor, which recognizes a base sequence of DNA as a target by the PPR protein part, and then controls transcription of the target DNA.

[0136] The functional region having a transcription control function usable for the present invention may be a domain that activates transcription, or may be a domain that suppresses transcription. Examples of the transcription control domain include VP16, VP64, TA2, STAT-6, and p65. Such a complex comprising a PPR protein and a transcription control domain does not exist in the nature, and is novel.

[0137] Further, the complex obtainable according to the present invention may deliver a functional region in a living body or cell in a DNA sequence-specific manner, and allow it to function. It thereby makes it possible to perform modification or disruption in a DNA sequence-specific manner in a living body or cell, like protein complexes utilizing a zinc finger protein (Non-patent documents 1 and 2 mentioned above) or TAL effecter (Non-patent document 3 and Patent document 1 mentioned above), and thus it becomes possible to impart a novel function, i.e., function for cleavage of DNA and genome edition utilizing that function. Specifically, with a PPR protein comprising two or more PPR motifs that can bind with a specific base linked together, a specific DNA sequence can be recognized. Then, genome edition of the recognized DNA region can be realized by the functional region bound to the PPR protein using the function of the functional region.

[0138] Furthermore, by binding a drug to the PPR protein that binds to a DNA sequence in a DNA sequence-specific manner, the drug may be delivered to the neighborhood of the DNA sequence as the target. Therefore, the present invention provides a method for DNA sequence-specific delivery of a functional substance.

[0139] It has been clarified that the PPR protein used as a material in the present invention works to specify an edition position for DNA edition, and such a PPR motif having specific amino acids arranged at the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. recognizes a specific base on DNA, and then exhibits the DNA-binding activity thereof. On the basis of such a characteristic, a PPR protein of this type that has specific amino acids arranged at the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. can be expected to recognize a base on DNA specific to each PPR protein, and as a result, introduce base polymorphism, or to be used in a treatment of a disease or condition resulting from a base polymorphism, and in addition, it is considered that the combination of such a PPR protein with such another functional region as mentioned above contribute to modification or improvement of functions for realizing cleavage of DNA for genome edition.

[0140] Moreover, an exogenous DNA-cleaving enzyme can be fused to the C-terminus of the PPR protein. Alternatively, by improving binding DNA base selectivity of the PPR motif on the N-terminus side, a DNA sequence-specific DNA-cleaving enzyme can also be constituted. Moreover, such a complex to which a marker part such as GFP is bound can also be used for visualization of a desired DNA in vivo.

EXAMPLES

Example 1: Collection of PPR Proteins and Target Sequences Thereof Used for DNA Edition

[0141] By referring to the information provided in the prior art references (Non-patent documents 11 to 15), structures and functions of the p63 protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQ ID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5) were analyzed.

[0142] To the PPR motif structures in such proteins, amino acid numbers defined in the present invention were imparted together with the information of the Uniprot database (http://www.uniprot.org/). The PPR motifs contained in the five kinds of PPR proteins of Arabidopsis thaliana (SEQ ID NOS: 1 to 5) used for the experiment, and the amino acid numbers thereof are shown in FIG. 3.

[0143] Specifically, amino acid frequencies for the amino acids at the three positions (No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A.) responsible for the nucleic acid recognition codes in the PPR motifs considered to be important at the time of targeting RNA in the aforementioned p63 protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQ ID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5) were compared with those of RNA-binding type motifs.

[0144] The p63 protein of Arabidopsis thaliana (SEQ ID NO: 1) has 9 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. in the amino acid sequence are as summarized in the following table and FIG. 3.

TABLE-US-00001 TABLE 1 Code Base to be bound (ratio) A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 230, V 233, R 263, S *R* 0.25 0.07 0.06 0.62 motif 1 PPR 265, F 268, D 297, S *D* 0.25 0.24 0.23 0.29 motif 2 PPR 299, L 302, K 322, D *KD 0.20 0.18 0.28 0.34 motif 3 PPR 334, Q 377, A 367, N *AN 0.45 0.18 0.05 0.32 motif 4 PPR 369, R 372, K 399, Y *K* 0.17 0.32 0.23 0.29 motif 5 PPR 401, E 404, L 434, S *LS 0.22 0.37 0.06 0.34 motif 6 PPR 436, S 439, S 469, E *SE 0.58 0.07 0.10 0.25 motif 7 PPR 471, T 474, D 505, M *D* 0.25 0.24 0.23 0.29 motif 8 PPR 507, N 510, M 540, R *M* 0.13 0.14 0.22 0.51 motif 9

[0145] The GUN1 protein of Arabidopsis thaliana (SEQ ID NO: 2) has 11 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. in the amino acid sequence are as summarized in the following table and FIG. 3.

TABLE-US-00002 TABLE 2 Code Base to be bound (ratio) A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 234, K 237, S 267, T *S* 0.41 0.12 0.22 0.25 motif 1 PPR 269, Y 272, S 302, N *SN 0.62 0.07 0.04 0.26 motif 2 PPR 304, V 307, N 338, D VND 0.06 0.21 0.06 0.66 motif 3 PPR 340, I 343, N 373, D IND 0.14 0.24 0.12 0.50 motif 4 PPR 375, F 378, N 408, N FNN 0.24 0.21 0.24 0.31 motif 5 PPR 410, V 413, S 443, D VSD 0.33 0.24 0.23 0.20 motif 6 PPR 445, V 448, N 478, D VND 0.06 0.21 0.06 0.66 motif 7 PPR 480, V 483, N 513, N VNN 0.17 0.48 0.09 0.26 motif 8 PPR 515, L 518, S 548, D *SD 0.20 0.17 0.39 0.24 motif 9 PPR 550, V 553, S 583, N VSN 0.57 0.09 0.05 0.30 motif 10 PPR 585, V 588, N 620, A *N* 0.10 0.33 0.10 0.48 motif 11

[0146] The pTac2 protein of Arabidopsis thaliana (SEQ ID NO: 3) has 15 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. in the amino acid sequence are as summarized in the following table and FIG. 3.

TABLE-US-00003 TABLE 3 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 106, N 109, A 140, N *AN 0.45 0.18 0.05 0.32 motif 1 PPR 142, H 145, T 175, S *TS 0.37 0.29 0.15 0.19 motif 2 PPR 177, F 180, T 210, S *TS 0.37 0.29 0.15 0.19 motif 3 PPR 212, L 215, N 246, D LND 0.08 0.15 0.23 0.54 motif 4 PPR 248, V 251, N 281, D VND 0.06 0.21 0.06 0.66 motif 5 PPR 283, T 286, S 316, D TSD 0.14 0.18 0.14 0.54 motif 6 PPR 318, T 321, N 351, N TNN 0.08 0.49 0.17 0.26 motif 7 PPR 353, N 356, S 386, D *SD 0.20 0.17 0.39 0.24 motif 8 PPR 388, A 491, N 421, D AND 0.07 0.05 0.14 0.74 motif 9 PPR 423, E 426, E 456, S B.G. 0.25 0.21 0.18 0.36 motif 10 PPR 458, K 461, T 491, S *TS 0.37 0.29 0.15 0.19 motif 11 PPR 493, E 496, H 526, N *H* 0.17 0.34 0.06 0.43 motif 12 PPR 528, D 531, N 561, D *ND 0.11 0.17 0.10 0.62 motif 13 PPR 563, R 566, E 596, S B.G. 0.25 0.21 0.18 0.36 motif 14 PPR 598, M 601, C 631. I *C* 0.55 0.10 0.21 0.14 motif 15 (B.G. means background)

[0147] The DG1 protein of Arabidopsis thaliana (SEQ ID NO: 4) has 10 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. in the amino acid sequence are as summarized in the following table and FIG. 3.

TABLE-US-00004 TABLE 4 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 256, F 259, T 290, D *TD 0.10 0.10 0.67 0.13 motif 1 PPR 292, A 295, H 340, D *H* 0.17 0.34 0.06 0.43 motif 2 PPR 342, V 345, N 375, N VNN 0.17 0.48 0.09 0.26 motif 3 PPR 377, A 380, G 410, K *G* 0.29 0.13 0.31 0.27 motif 4 PPR 412, I 415, K 445, T *K* 0.17 0.32 0.23 0.29 motif 5 PPR 477, S 450, Y 481, L B.G. 0.25 0.21 0.18 0.36 motif 6 PPR 483, I 486, T 515, N ITN 0.79 0.06 0.05 0.10 motif 7 PPR 517, G 520, N 553, N *NN 0.12 0.44 0.13 0.30 motif 8 PPR 555, Y 558, S 588, D YSD 0.25 0.15 0.39 0.20 motif 9 PPR 590, T 593, A 623, H *AH 0.41 0.08 0.07 0.45 motif 10 (B.G. means background)

[0148] The GRP23 protein of Arabidopsis thaliana (SEQ ID NO: 5) has 11 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. "ii" (-2) A.A. in the amino acid sequence are as summarized in the following table and FIG. 3.

TABLE-US-00005 TABLE 5 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 181, F 184, N 215, N FNN 0.24 0.21 0.24 0.31 motif 1 PPR 217, V 220, N 251, S VNS 0.07 0.61 0.05 0.27 motif 2 PPR 253, V 256, R 286, D *RD 0.25 0.07 0.06 0.62 motif 3 PPR 288, T 291, N 321, D TND 0.14 0.08 0.07 0.71 motif 4 PPR 323, I 326, A 356, H *AH 0.41 0.08 0.07 0.45 motif 5 PPR 358, P 361, N 396, N *NN 0.12 0.44 0.13 0.30 motif 6 PPR 398, D 401, G 435, D *GD 0.09 0.09 0.59 0.25 motif 7 PPR 437, L 404, C 470, D *CD 0.30 0.15 0.35 0.20 motif 8 PPR 472, P 475, R 505, V *R* 0.25 0.07 0.06 0.62 motif 9 PPR 507, D 510, A 540, D *AD 0.10 0.22 0.39 0.29 motif 10 PPR 542, S 545, D 575, T *D* 0.25 0.24 0.23 0.29 motif 11 (B.G. means background)

[0149] The amino acid frequencies for these positions were confirmed for each protein, and compared with the amino acid frequencies for the same positions of the RNA-binding type motifs. The results are shown in FIG. 2. It became clear that the tendencies of the amino acid frequencies in the PPR motifs of the PPR proteins for which DNA-binding property is suggested, and the RNA-binding type motifs substantially agreed with each other. That is, it became clear that the PPR proteins that act to bind to DNA bind with nucleic acids according to same sequence rules as those of the PPR proteins that act to bind to RNA, and the RNA recognition codes described in the pending patent application of the inventors of the present invention (PCT/JP2012/077274) can be applied as the DNA recognition codes of the PPR proteins that act to bind to DNA.

[0150] With reference to the RNA recognition codes described in the non-patent document (Yagi, Y. et al., Plos One, 2013, 8, e57286), the DNA-binding type PPR motifs that selectively bind to each corresponding base were evaluated. More precisely, a chi square test was performed on the basis of occurrence nucleotide frequencies shown in Table 6 and expected nucleotide frequencies calculated from the background frequencies. The test was performed for each base (NT), purine or pyrimidine (AG or CT, PY), hydrogen bond group (AT or GC, HB), or amino or keto form (AC or GT). Significant value was defined as P<0.06 (5E-02, 5% significance level), and when a significant value was obtained in any of the tests, the combination of No. 1 amino acid, No. 4 amino acid, and No. "ii" (-2) amino acid was chosen.

TABLE-US-00006 TABLE 6 Base selectivity of DNA-binding code NSRs occurrence of the Probabilitiy matrix Subtraction for background (1, 4, ii) NSR(s) A C G T A C G T *GD 14 0.10 0.06 0.57 0.28 -0.16 -0.15 0.40 -0.08 EGD 8 0.07 0.05 0.69 0.19 -0.19 -0.16 0.52 -0.17 *GN 11 0.55 0.10 0.04 0.31 0.29 -0.11 -0.13 -0.05 EGN 5 0.63 0.06 0.05 0.25 0.37 -0.15 -0.12 -0.11 *GS 3 0.57 0.23 0.06 0.14 0.31 0.02 -0.11 -0.22 *I* 15 0.15 0.29 0.10 0.45 -0.11 0.08 -0.07 0.09 *IN 4 0.17 0.28 0.06 0.50 -0.09 0.07 -0.11 0.14 *L* 23 0.20 0.30 0.03 0.47 -0.06 0.09 -0.14 0.11 *LD 5 0.19 0.47 0.05 0.28 -0.07 0.26 -0.12 -0.08 *LK 3 0.09 0.08 0.06 0.77 -0.17 -0.13 -0.11 0.41 *M* 10 0.14 0.15 0.15 0.56 -0.12 -0.06 -0.02 0.20 *MD 9 0.15 0.13 0.17 0.55 -0.11 -0.08 0.00 0.19 IMD 4 0.09 0.24 0.06 0.62 -0.17 0.03 -0.11 0.26 *N* 147 0.11 0.33 0.10 0.45 -0.15 0.12 -0.07 0.09 ND 72 0.11 0.18 0.10 0.61 -0.15 -0.03 -0.07 0.25 FND 13 0.23 0.19 0.10 0.49 -0.03 -0.02 -0.07 0.13 GND 3 0.09 0.08 0.06 0.77 -0.17 -0.13 -0.11 0.41 IND 5 0.22 0.13 0.05 0.60 -0.04 -0.08 -0.12 0.24 TND 3 0.15 0.08 0.06 0.72 -0.11 -0.13 -0.11 0.36 VND 23 0.06 0.25 0.06 0.63 -0.20 0.04 -0.11 0.27 YND 6 0.08 0.30 0.11 0.52 -0.18 0.09 -0.06 0.16 *NN 34 0.15 0.45 0.14 0.27 -0.11 0.24 -0.03 -0.09 INN 7 0.12 0.49 0.05 0.34 -0.14 0.28 -0.12 -0.02 SNN 3 0.09 0.60 0.06 0.24 -0.17 0.39 -0.11 -0.12 VNN 10 0.20 0.53 0.04 0.23 -0.06 0.32 -0.13 -0.13 *NS 13 0.11 0.47 0.07 0.36 -0.15 0.26 -0.10 0.00 VNS 5 0.08 0.66 0.05 0.21 -0.18 0.45 -0.12 -0.15 *NT 13 0.12 0.52 0.13 0.24 -0.14 0.31 -0.04 -0.12 VNT 5 0.08 0.57 0.05 0.30 -0.18 0.36 -0.12 -0.06 *NW 11 0.14 0.32 0.13 0.41 -0.12 0.11 -0.04 0.05 INW 3 0.09 0.29 0.06 0.56 -0.17 0.08 -0.11 0.20 *P* 17 0.10 0.06 0.11 0.73 -0.16 -0.15 -0.06 0.37 *PD 9 0.06 0.09 0.10 0.75 -0.20 -0.12 -0.07 0.39 FPD 3 0.09 0.08 0.06 0.77 -0.17 -0.13 -0.11 0.41 YPD 3 0.09 0.08 0.06 0.77 -0.17 -0.13 -0.11 0.41 *S* 49 0.38 0.13 0.20 0.29 0.12 -0.08 0.03 -0.07 *SN 18 0.63 0.08 0.05 0.24 0.37 -0.13 -0.12 -0.12 FSN 7 0.63 0.13 0.08 0.16 0.37 -0.08 -0.09 -0.20 VSN 6 0.60 0.10 0.05 0.25 0.34 -0.11 -0.12 -0.11 *T* 86 0.45 0.09 0.31 0.15 0.19 -0.12 0.14 -0.21 *TD 32 0.13 0.12 0.61 0.14 -0.13 -0.09 0.44 -0.22 VTD 7 0.07 0.06 0.67 0.20 -0.19 -0.15 0.50 -0.16 *TN 31 0.66 0.08 0.13 0.13 0.40 -0.13 -0.04 -0.23 FTN 4 0.75 0.07 0.06 0.12 0.49 -0.14 -0.11 -0.24 ITN 5 0.77 0.06 0.05 0.11 0.51 -0.15 -0.12 -0.25 VTN 10 0.63 0.13 0.15 0.09 0.37 -0.08 -0.02 -0.27 *V* 48 0.29 0.21 0.08 0.43 0.03 0.00 -0.09 0.07 IVD 3 0.31 0.50 0.06 0.14 0.05 0.29 -0.11 -0.22 *VG 5 0.22 0.48 0.05 0.25 -0.04 0.27 -0.12 -0.11 *VT 4 0.25 0.07 0.06 0.62 -0.01 -0.14 -0.11 0.26 Background frequency 0.26 0.21 0.17 0.36

[0151] In Table 1, the combinations of the amino acids that showed significant base selectivity were mentioned. That is, these results mean that the PPR motifs having the amino acid species of the No. 1 amino acid, No. 4 amino acid, and No. "ii" (-2) amino acid ("NSRs (1, 4, and ii)" in the table) that provided a significant P value are PPR motifs that impart base-selective binding ability, and a larger "positive" value obtained after the subtraction of the background means higher base selectivity for the base. Among the No. 1 amino acid, No. 4 amino acid, and No. "ii" (-2) amino acid, the No. 4 amino acid most strongly affects the base selectivity, the No. "ii" (-2) amino acid affects the base selectivity next strongly, and the No. 1 amino acid most weakly affects the base selectivity among the three amino acids.

Example 2: Evaluation of Sequence-Specific DNA-Binding Ability PPR Molecules

[0152] In this example, artificial transcription factors were prepared by fusing VP64, which is a transcription activation domain, to the three kinds of DNA-binding type (expectedly) PPR molecules, p63, pTac2, and GUN1, and by examining whether they could activate luciferase reporters each having a corresponding target sequence in a human cultured cell, whether the PPR molecules had a sequence-specific DNA-binding ability or not was determined (FIG. 5).

Experimental Method

[0153] 1. Preparation of PPR-VP64 expression vector

[0154] Only the parts corresponding to the PPR motifs in the coding sequences of p63, pTac2, and GUN1 were prepared by artificial synthesis. For the DNA synthesis, the artificial gene synthesis service of Biomatik was used. The pCS2P vector having the CMV promoter was used as a backbone vector, and each synthesized PPR sequence was inserted into it. Further, the Flag tag and nuclear transfer signal were inserted at the N-terminus of the PPR sequence, and the VP64 sequence was inserted at the C-terminus of the same. The produced sequences of p63-VP64, pTac2-VP64, and GUN1-VP64 are shown in Sequence Listing as SEQ ID NOS: 7 to 9.

[0155] 2. Preparation of Reporter Vector Having PPR Target Sequence

[0156] A reporter vector (pminCMV-luc2, SEQ ID NO: 10) was prepared, in which the firefly luciferase gene was ligated downstream from the Minimal CMV promoter, and a multi-cloning site was placed upstream of the promoter. The predicted target sequence of each PPR was inserted into the vector at the multi-cloning site. The target sequence of each PPR (TCTATCACT for p63, AACTTTCGTCACTCA for pTac2, and AATTTGTCGAT for GUN1, SEQ ID NOS: 11 to 13 in Sequence Listing) was determined by predicting the motif-DNA recognition codes of DNA-binding type PPR from the motif-RNA recognition codes observed in the RNA-binding type PPR. For each PPR, sequences containing 4 or 8 of target sequences were prepared, and used in the following assay. The nucleotide sequences of the vectors are shown as SEQ ID NOS: 14 to 19 in Sequence Listing.

[0157] 3. Transfection into HEK293 T Cell

[0158] The PPR-VP64 expression vector prepared in the section 1, the firefly luciferase expression vector prepared in the section 2, and the pRL-CMV vector (expression vector for Renilla luciferase, Promega) as a reference were introduced by using Lipofectamine LTX (Life Technologies). The DMEM medium (25 .mu.l) was added to each well of a 96-well plate, and a mixture containing the PPR-VP64 expression vector (400 ng), firefly luciferase expression vector (100 ng), and pRL-CMV vector (20 ng) was further added. Then, a mixture of the DMEM medium (25 .mu.l) and Lipofectamine LTX (0.7 .mu.l) was added to each well, the plate was left standing at room temperature for 30 minutes, then 6.times.10.sup.4 of the HEK293 T cells suspended in the DMEM medium containing 15% fetal bovine serum (100 .mu.l) were added, and the cells were cultured at 37.degree. C. in a CO2 incubator for 24 hours.

[0159] 4. Luciferase Assay

[0160] Luciferase assay was performed by using Dual-Glo Luciferase Assay System (Promega) in accordance with the instructions attached to the kit. For the measurement of the luciferase activity, Tri Star LB 941 Plate Reader (Berthold) was used.

[0161] (Results and Discussion)

[0162] The luciferase activity was compared for the cases of introducing pTac2-VP64 or GUN1-VP64 together with pminCMV-luc2 for a negative control, or the reporter vector having 4 or 8 target sequences (table mentioned below, FIG. 6). The comparison of the activity was performed on the basis of standardized scores obtained by dividing the measured values obtained with Fluc (firefly luciferase) with the measured value obtained with Rluc (Renilla luciferase) as the reference (Fluc/Rluc). As a result, there was observed a tendency that the activity increased with increase of the number of the target sequence for the both cases, and thus it was verified that each of the PPR-VP64 molecules specifically bound to each target sequence, and functioned as a site-specific transcription activator.

TABLE-US-00007 Fluc reporter PPR-VP64 Reference Fluc Rluc Fluc/Rluc Fold activation pTac2-VP64 (negative control) pminCMV-luc2 pTac2-VP64 pRL-CMV 47744 4948 9.649151172 1 pTac2-VP64 (4x target) pTac2-4x target pTac2-VP64 pRL-CMV 133465 4757 28.05654824 2.907670089 pTac2-VP64 (8x target) pTac2-8x target pTac2-VP64 pRL-CMV 189146 4011 47.15681875 4.887146849 GUN1-VP64 (negative control) pminCMV-luc2 GUN1-VP64 pRL-CMV 29590 3799 7.788891814 1 GUN1-VP64 (4x target) GUN1-4x target GUN1-VP64 pRL-CMV 61070 2727 22.39457279 2.875193715 GUN1-VP64 (8x target) GUN1-8x target GUN1-VP64 pRL-CMV 66982 2731 24.52654705 3.14891356

Sequence CWU 1

1

231596PRTArabidopsis thaliana 1Met Phe Ala Leu Ser Lys Val Leu Arg Arg Thr Gln Arg Leu Arg Leu1 5 10 15Gly Ala Cys Ser Ala Val Phe Ser Lys Asp Ile Gln Leu Gly Gly Glu 20 25 30Arg Ser Phe Asp Ser Asn Ser Ile Ala Ser Thr Lys Arg Glu Ala Val 35 40 45Pro Arg Phe Tyr Glu Ile Ser Ser Leu Ser Asn Arg Ala Leu Ser Ser 50 55 60Ser Ala Gly Thr Lys Ser Asp Gln Glu Glu Asp Asp Leu Glu Asp Gly65 70 75 80Phe Ser Glu Leu Glu Gly Ser Lys Ser Gly Gln Gly Ser Thr Ser Ser 85 90 95Asp Glu Asp Glu Gly Lys Leu Ser Ala Asp Glu Glu Glu Glu Glu Glu 100 105 110Leu Asp Leu Ile Glu Thr Asp Val Ser Arg Lys Thr Val Glu Lys Lys 115 120 125Gln Ser Glu Leu Phe Lys Thr Ile Val Ser Ala Pro Gly Leu Ser Ile 130 135 140Gly Ser Ala Leu Asp Lys Trp Val Glu Glu Gly Asn Glu Ile Thr Arg145 150 155 160Val Glu Ile Ala Lys Ala Met Leu Gln Leu Arg Arg Arg Arg Met Tyr 165 170 175Gly Arg Ala Leu Gln Met Ser Glu Trp Leu Glu Ala Asn Lys Lys Ile 180 185 190Glu Met Thr Glu Arg Asp Tyr Ala Ser Arg Leu Asp Leu Thr Val Lys 195 200 205Ile Arg Gly Leu Glu Lys Gly Glu Ala Cys Met Gln Lys Ile Pro Lys 210 215 220Ser Phe Lys Gly Glu Val Leu Tyr Arg Thr Leu Leu Ala Asn Cys Val225 230 235 240Ala Ala Gly Asn Val Lys Lys Ser Glu Leu Val Phe Asn Lys Met Lys 245 250 255Asp Leu Gly Phe Pro Leu Ser Gly Phe Thr Cys Asp Gln Met Leu Leu 260 265 270Leu His Lys Arg Ile Asp Arg Lys Lys Ile Ala Asp Val Leu Leu Leu 275 280 285Met Glu Lys Glu Asn Ile Lys Pro Ser Leu Leu Thr Tyr Lys Ile Leu 290 295 300Ile Asp Val Lys Gly Ala Thr Asn Asp Ile Ser Gly Met Glu Gln Ile305 310 315 320Leu Glu Thr Met Lys Asp Glu Gly Val Glu Leu Asp Phe Gln Thr Gln 325 330 335Ala Leu Thr Ala Arg His Tyr Ser Gly Ala Gly Leu Lys Asp Lys Ala 340 345 350Glu Lys Val Leu Lys Glu Met Glu Gly Glu Ser Leu Glu Ala Asn Arg 355 360 365Arg Ala Phe Lys Asp Leu Leu Ser Ile Tyr Ala Ser Leu Gly Arg Glu 370 375 380Asp Glu Val Lys Arg Ile Trp Lys Ile Cys Glu Ser Lys Pro Tyr Phe385 390 395 400Glu Glu Ser Leu Ala Ala Ile Gln Ala Phe Gly Lys Leu Asn Lys Val 405 410 415Gln Glu Ala Glu Ala Ile Phe Glu Lys Ile Val Lys Met Asp Arg Arg 420 425 430Ala Ser Ser Ser Thr Tyr Ser Val Leu Leu Arg Val Tyr Val Asp His 435 440 445Lys Met Leu Ser Lys Gly Lys Asp Leu Val Lys Arg Met Ala Glu Ser 450 455 460Gly Cys Arg Ile Glu Ala Thr Thr Trp Asp Ala Leu Ile Lys Leu Tyr465 470 475 480Val Glu Ala Gly Glu Val Glu Lys Ala Asp Ser Leu Leu Asp Lys Ala 485 490 495Ser Lys Gln Ser His Thr Lys Leu Met Met Asn Ser Phe Met Tyr Ile 500 505 510Met Asp Glu Tyr Ser Lys Arg Gly Asp Val His Asn Thr Glu Lys Ile 515 520 525Phe Leu Lys Met Arg Glu Ala Gly Tyr Thr Ser Arg Leu Arg Gln Phe 530 535 540Gln Ala Leu Met Gln Ala Tyr Ile Asn Ala Lys Ser Pro Ala Tyr Gly545 550 555 560Met Arg Asp Arg Leu Lys Ala Asp Asn Ile Phe Pro Asn Lys Ser Met 565 570 575Ala Ala Gln Leu Ala Gln Gly Asp Pro Phe Lys Lys Thr Ala Ile Ser 580 585 590Asp Ile Leu Asp 5952918PRTArabidopsis thaliana 2Met Ala Ser Thr Pro Pro His Trp Val Thr Thr Thr Asn Asn His Arg1 5 10 15Pro Trp Leu Pro Gln Arg Pro Arg Pro Gly Arg Ser Val Thr Ser Ala 20 25 30Pro Pro Ser Ser Ser Ala Ser Val Ser Ser Ala His Leu Ser Gln Thr 35 40 45Thr Pro Asn Phe Ser Pro Leu Gln Thr Pro Lys Ser Asp Phe Ser Gly 50 55 60Arg Gln Ser Thr Arg Phe Val Ser Pro Ala Thr Asn Asn His Arg Gln65 70 75 80Thr Arg Gln Asn Pro Asn Tyr Asn His Arg Pro Tyr Gly Ala Ser Ser 85 90 95Ser Pro Arg Gly Ser Ala Pro Pro Pro Ser Ser Val Ala Thr Val Ala 100 105 110Pro Ala Gln Leu Ser Gln Pro Pro Asn Phe Ser Pro Leu Gln Thr Pro 115 120 125Lys Ser Asp Leu Ser Ser Asp Phe Ser Gly Arg Arg Ser Thr Arg Phe 130 135 140Val Ser Lys Met His Phe Gly Arg Gln Lys Thr Thr Met Ala Thr Arg145 150 155 160His Ser Ser Ala Ala Glu Asp Ala Leu Gln Asn Ala Ile Asp Phe Ser 165 170 175Gly Asp Asp Glu Met Phe His Ser Leu Met Leu Ser Phe Glu Ser Lys 180 185 190Leu Cys Gly Ser Asp Asp Cys Thr Tyr Ile Ile Arg Glu Leu Gly Asn 195 200 205Arg Asn Glu Cys Asp Lys Ala Val Gly Phe Tyr Glu Phe Ala Val Lys 210 215 220Arg Glu Arg Arg Lys Asn Glu Gln Gly Lys Leu Ala Ser Ala Met Ile225 230 235 240Ser Thr Leu Gly Arg Tyr Gly Lys Val Thr Ile Ala Lys Arg Ile Phe 245 250 255Glu Thr Ala Phe Ala Gly Gly Tyr Gly Asn Thr Val Tyr Ala Phe Ser 260 265 270Ala Leu Ile Ser Ala Tyr Gly Arg Ser Gly Leu His Glu Glu Ala Ile 275 280 285Ser Val Phe Asn Ser Met Lys Glu Tyr Gly Leu Arg Pro Asn Leu Val 290 295 300Thr Tyr Asn Ala Val Ile Asp Ala Cys Gly Lys Gly Gly Met Glu Phe305 310 315 320Lys Gln Val Ala Lys Phe Phe Asp Glu Met Gln Arg Asn Gly Val Gln 325 330 335Pro Asp Arg Ile Thr Phe Asn Ser Leu Leu Ala Val Cys Ser Arg Gly 340 345 350Gly Leu Trp Glu Ala Ala Arg Asn Leu Phe Asp Glu Met Thr Asn Arg 355 360 365Arg Ile Glu Gln Asp Val Phe Ser Tyr Asn Thr Leu Leu Asp Ala Ile 370 375 380Cys Lys Gly Gly Gln Met Asp Leu Ala Phe Glu Ile Leu Ala Gln Met385 390 395 400Pro Val Lys Arg Ile Met Pro Asn Val Val Ser Tyr Ser Thr Val Ile 405 410 415Asp Gly Phe Ala Lys Ala Gly Arg Phe Asp Glu Ala Leu Asn Leu Phe 420 425 430Gly Glu Met Arg Tyr Leu Gly Ile Ala Leu Asp Arg Val Ser Tyr Asn 435 440 445Thr Leu Leu Ser Ile Tyr Thr Lys Val Gly Arg Ser Glu Glu Ala Leu 450 455 460Asp Ile Leu Arg Glu Met Ala Ser Val Gly Ile Lys Lys Asp Val Val465 470 475 480Thr Tyr Asn Ala Leu Leu Gly Gly Tyr Gly Lys Gln Gly Lys Tyr Asp 485 490 495Glu Val Lys Lys Val Phe Thr Glu Met Lys Arg Glu His Val Leu Pro 500 505 510Asn Leu Leu Thr Tyr Ser Thr Leu Ile Asp Gly Tyr Ser Lys Gly Gly 515 520 525Leu Tyr Lys Glu Ala Met Glu Ile Phe Arg Glu Phe Lys Ser Ala Gly 530 535 540Leu Arg Ala Asp Val Val Leu Tyr Ser Ala Leu Ile Asp Ala Leu Cys545 550 555 560Lys Asn Gly Leu Val Gly Ser Ala Val Ser Leu Ile Asp Glu Met Thr 565 570 575Lys Glu Gly Ile Ser Pro Asn Val Val Thr Tyr Asn Ser Ile Ile Asp 580 585 590Ala Phe Gly Arg Ser Ala Thr Met Asp Arg Ser Ala Asp Tyr Ser Asn 595 600 605Gly Gly Ser Leu Pro Phe Ser Ser Ser Ala Leu Ser Ala Leu Thr Glu 610 615 620Thr Glu Gly Asn Arg Val Ile Gln Leu Phe Gly Gln Leu Thr Thr Glu625 630 635 640Ser Asn Asn Arg Thr Thr Lys Asp Cys Glu Glu Gly Met Gln Glu Leu 645 650 655Ser Cys Ile Leu Glu Val Phe Arg Lys Met His Gln Leu Glu Ile Lys 660 665 670Pro Asn Val Val Thr Phe Ser Ala Ile Leu Asn Ala Cys Ser Arg Cys 675 680 685Asn Ser Phe Glu Asp Ala Ser Met Leu Leu Glu Glu Leu Arg Leu Phe 690 695 700Asp Asn Lys Val Tyr Gly Val Val His Gly Leu Leu Met Gly Gln Arg705 710 715 720Glu Asn Val Trp Leu Gln Ala Gln Ser Leu Phe Asp Lys Val Asn Glu 725 730 735Met Asp Gly Ser Thr Ala Ser Ala Phe Tyr Asn Ala Leu Thr Asp Met 740 745 750Leu Trp His Phe Gly Gln Lys Arg Gly Ala Glu Leu Val Ala Leu Glu 755 760 765Gly Arg Ser Arg Gln Val Trp Glu Asn Val Trp Ser Asp Ser Cys Leu 770 775 780Asp Leu His Leu Met Ser Ser Gly Ala Ala Arg Ala Met Val His Ala785 790 795 800Trp Leu Leu Asn Ile Arg Ser Ile Val Tyr Glu Gly His Glu Leu Pro 805 810 815Lys Val Leu Ser Ile Leu Thr Gly Trp Gly Lys His Ser Lys Val Val 820 825 830Gly Asp Gly Ala Leu Arg Arg Ala Val Glu Val Leu Leu Arg Gly Met 835 840 845Asp Ala Pro Phe His Leu Ser Lys Cys Asn Met Gly Arg Phe Thr Ser 850 855 860Ser Gly Ser Val Val Ala Thr Trp Leu Arg Glu Ser Ala Thr Leu Lys865 870 875 880Leu Leu Ile Leu His Asp His Ile Thr Thr Ala Thr Ala Thr Thr Thr 885 890 895Thr Met Lys Ser Thr Asp Gln Gln Gln Arg Lys Gln Thr Ser Phe Ala 900 905 910Leu Gln Pro Leu Leu Leu 9153862PRTArabidopsis thaliana 3Met Asn Leu Ala Ile Pro Asn Pro Asn Ser His His Leu Ser Phe Leu1 5 10 15Ile Gln Asn Ser Ser Phe Ile Gly Asn Arg Arg Phe Ala Asp Gly Asn 20 25 30Arg Leu Arg Phe Leu Ser Gly Gly Asn Arg Lys Pro Cys Ser Phe Ser 35 40 45Gly Lys Ile Lys Ala Lys Thr Lys Asp Leu Val Leu Gly Asn Pro Ser 50 55 60Val Ser Val Glu Lys Gly Lys Tyr Ser Tyr Asp Val Glu Ser Leu Ile65 70 75 80Asn Lys Leu Ser Ser Leu Pro Pro Arg Gly Ser Ile Ala Arg Cys Leu 85 90 95Asp Ile Phe Lys Asn Lys Leu Ser Leu Asn Asp Phe Ala Leu Val Phe 100 105 110Lys Glu Phe Ala Gly Arg Gly Asp Trp Gln Arg Ser Leu Arg Leu Phe 115 120 125Lys Tyr Met Gln Arg Gln Ile Trp Cys Lys Pro Asn Glu His Ile Tyr 130 135 140Thr Ile Met Ile Ser Leu Leu Gly Arg Glu Gly Leu Leu Asp Lys Cys145 150 155 160Leu Glu Val Phe Asp Glu Met Pro Ser Gln Gly Val Ser Arg Ser Val 165 170 175Phe Ser Tyr Thr Ala Leu Ile Asn Ala Tyr Gly Arg Asn Gly Arg Tyr 180 185 190Glu Thr Ser Leu Glu Leu Leu Asp Arg Met Lys Asn Glu Lys Ile Ser 195 200 205Pro Ser Ile Leu Thr Tyr Asn Thr Val Ile Asn Ala Cys Ala Arg Gly 210 215 220Gly Leu Asp Trp Glu Gly Leu Leu Gly Leu Phe Ala Glu Met Arg His225 230 235 240Glu Gly Ile Gln Pro Asp Ile Val Thr Tyr Asn Thr Leu Leu Ser Ala 245 250 255Cys Ala Ile Arg Gly Leu Gly Asp Glu Ala Glu Met Val Phe Arg Thr 260 265 270Met Asn Asp Gly Gly Ile Val Pro Asp Leu Thr Thr Tyr Ser His Leu 275 280 285Val Glu Thr Phe Gly Lys Leu Arg Arg Leu Glu Lys Val Cys Asp Leu 290 295 300Leu Gly Glu Met Ala Ser Gly Gly Ser Leu Pro Asp Ile Thr Ser Tyr305 310 315 320Asn Val Leu Leu Glu Ala Tyr Ala Lys Ser Gly Ser Ile Lys Glu Ala 325 330 335Met Gly Val Phe His Gln Met Gln Ala Ala Gly Cys Thr Pro Asn Ala 340 345 350Asn Thr Tyr Ser Val Leu Leu Asn Leu Phe Gly Gln Ser Gly Arg Tyr 355 360 365Asp Asp Val Arg Gln Leu Phe Leu Glu Met Lys Ser Ser Asn Thr Asp 370 375 380Pro Asp Ala Ala Thr Tyr Asn Ile Leu Ile Glu Val Phe Gly Glu Gly385 390 395 400Gly Tyr Phe Lys Glu Val Val Thr Leu Phe His Asp Met Val Glu Glu 405 410 415Asn Ile Glu Pro Asp Met Glu Thr Tyr Glu Gly Ile Ile Phe Ala Cys 420 425 430Gly Lys Gly Gly Leu His Glu Asp Ala Arg Lys Ile Leu Gln Tyr Met 435 440 445Thr Ala Asn Asp Ile Val Pro Ser Ser Lys Ala Tyr Thr Gly Val Ile 450 455 460Glu Ala Phe Gly Gln Ala Ala Leu Tyr Glu Glu Ala Leu Val Ala Phe465 470 475 480Asn Thr Met His Glu Val Gly Ser Asn Pro Ser Ile Glu Thr Phe His 485 490 495Ser Leu Leu Tyr Ser Phe Ala Arg Gly Gly Leu Val Lys Glu Ser Glu 500 505 510Ala Ile Leu Ser Arg Leu Val Asp Ser Gly Ile Pro Arg Asn Arg Asp 515 520 525Thr Phe Asn Ala Gln Ile Glu Ala Tyr Lys Gln Gly Gly Lys Phe Glu 530 535 540Glu Ala Val Lys Thr Tyr Val Asp Met Glu Lys Ser Arg Cys Asp Pro545 550 555 560Asp Glu Arg Thr Leu Glu Ala Val Leu Ser Val Tyr Ser Phe Ala Arg 565 570 575Leu Val Asp Glu Cys Arg Glu Gln Phe Glu Glu Met Lys Ala Ser Asp 580 585 590Ile Leu Pro Ser Ile Met Cys Tyr Cys Met Met Leu Ala Val Tyr Gly 595 600 605Lys Thr Glu Arg Trp Asp Asp Val Asn Glu Leu Leu Glu Glu Met Leu 610 615 620Ser Asn Arg Val Ser Asn Ile His Gln Val Ile Gly Gln Met Ile Lys625 630 635 640Gly Asp Tyr Asp Asp Asp Ser Asn Trp Gln Ile Val Glu Tyr Val Leu 645 650 655Asp Lys Leu Asn Ser Glu Gly Cys Gly Leu Gly Ile Arg Phe Tyr Asn 660 665 670Ala Leu Leu Asp Ala Leu Trp Trp Leu Gly Gln Lys Glu Arg Ala Ala 675 680 685Arg Val Leu Asn Glu Ala Thr Lys Arg Gly Leu Phe Pro Glu Leu Phe 690 695 700Arg Lys Asn Lys Leu Val Trp Ser Val Asp Val His Arg Met Ser Glu705 710 715 720Gly Gly Met Tyr Thr Ala Leu Ser Val Trp Leu Asn Asp Ile Asn Asp 725 730 735Met Leu Leu Lys Gly Asp Leu Pro Gln Leu Ala Val Val Val Ser Val 740 745 750Arg Gly Gln Leu Glu Lys Ser Ser Ala Ala Arg Glu Ser Pro Ile Ala 755 760 765Lys Ala Ala Phe Ser Phe Leu Gln Asp His Val Ser Ser Ser Phe Ser 770 775 780Phe Thr Gly Trp Asn Gly Gly Arg Ile Met Cys Gln Arg Ser Gln Leu785 790 795 800Lys Gln Leu Leu Ser Thr Lys Glu Pro Thr Ser Glu Glu Ser Glu Asn 805 810 815Lys Asn Leu Val Ala Leu Ala Asn Ser Pro Ile Phe Ala Ala Gly Thr 820 825 830Arg Ala Ser Thr Ser Ser Asp Thr Asn His Ser Gly Asn Pro Thr Gln 835 840 845Arg Arg Thr Arg Thr Lys Lys Glu Leu Ala Gly Ser Thr Ala 850 855 8604798PRTArabidopsis thaliana 4Met Asp Ala Ser Val Val Arg Phe Ser Gln Ser Pro Ala Arg Val Pro1 5 10 15Pro Glu Phe Glu Pro Asp Met Glu Lys Ile Lys Arg Arg Leu Leu Lys 20 25 30Tyr Gly Val Asp Pro Thr Pro Lys Ile Leu Asn Asn Leu Arg Lys Lys 35 40 45Glu Ile Gln Lys His Asn Arg Arg Thr Lys Arg Glu Thr Glu Ser Glu 50 55 60Ala Glu Val Tyr Thr Glu Ala Gln Lys Gln Ser Met Glu Glu Glu Ala65 70 75 80Arg Phe Gln Thr Leu Arg Arg Glu Tyr Lys

Gln Phe Thr Arg Ser Ile 85 90 95Ser Gly Lys Arg Gly Gly Asp Val Gly Leu Met Val Gly Asn Pro Trp 100 105 110Glu Gly Ile Glu Arg Val Lys Leu Lys Glu Leu Val Ser Gly Val Arg 115 120 125Arg Glu Glu Val Ser Ala Gly Glu Leu Lys Lys Glu Asn Leu Lys Glu 130 135 140Leu Lys Lys Ile Leu Glu Lys Asp Leu Arg Trp Val Leu Asp Asp Asp145 150 155 160Val Asp Val Glu Glu Phe Asp Leu Asp Lys Glu Phe Asp Pro Ala Lys 165 170 175Arg Trp Arg Asn Glu Gly Glu Ala Val Arg Val Leu Val Asp Arg Leu 180 185 190Ser Gly Arg Glu Ile Asn Glu Lys His Trp Lys Phe Val Arg Met Met 195 200 205Asn Gln Ser Gly Leu Gln Phe Thr Glu Asp Gln Met Leu Lys Ile Val 210 215 220Asp Arg Leu Gly Arg Lys Gln Ser Trp Lys Gln Ala Ser Ala Val Val225 230 235 240His Trp Val Tyr Ser Asp Lys Lys Arg Lys His Leu Arg Ser Arg Phe 245 250 255Val Tyr Thr Lys Leu Leu Ser Val Leu Gly Phe Ala Arg Arg Pro Gln 260 265 270Glu Ala Leu Gln Ile Phe Asn Gln Met Leu Gly Asp Arg Gln Leu Tyr 275 280 285Pro Asp Met Ala Ala Tyr His Cys Ile Ala Val Thr Leu Gly Gln Ala 290 295 300Gly Leu Leu Lys Glu Leu Leu Lys Val Ile Glu Arg Met Arg Gln Lys305 310 315 320Pro Thr Lys Leu Thr Lys Asn Leu Arg Gln Lys Asn Trp Asp Pro Val 325 330 335Leu Glu Pro Asp Leu Val Val Tyr Asn Ala Ile Leu Asn Ala Cys Val 340 345 350Pro Thr Leu Gln Trp Lys Ala Val Ser Trp Val Phe Val Glu Leu Arg 355 360 365Lys Asn Gly Leu Arg Pro Asn Gly Ala Thr Tyr Gly Leu Ala Met Glu 370 375 380Val Met Leu Glu Ser Gly Lys Phe Asp Arg Val His Asp Phe Phe Arg385 390 395 400Lys Met Lys Ser Ser Gly Glu Ala Pro Lys Ala Ile Thr Tyr Lys Val 405 410 415Leu Val Arg Ala Leu Trp Arg Glu Gly Lys Ile Glu Glu Ala Val Glu 420 425 430Ala Val Arg Asp Met Glu Gln Lys Gly Val Ile Gly Thr Gly Ser Val 435 440 445Tyr Tyr Glu Leu Ala Cys Cys Leu Cys Asn Asn Gly Arg Trp Cys Asp 450 455 460Ala Met Leu Glu Val Gly Arg Met Lys Arg Leu Glu Asn Cys Arg Pro465 470 475 480Leu Glu Ile Thr Phe Thr Gly Leu Ile Ala Ala Ser Leu Asn Gly Gly 485 490 495His Val Asp Asp Cys Met Ala Ile Phe Gln Tyr Met Lys Asp Lys Cys 500 505 510Asp Pro Asn Ile Gly Thr Ala Asn Met Met Leu Lys Val Tyr Gly Arg 515 520 525Asn Asp Met Phe Ser Glu Ala Lys Glu Leu Phe Glu Glu Ile Val Ser 530 535 540Arg Lys Glu Thr His Leu Val Pro Asn Glu Tyr Thr Tyr Ser Phe Met545 550 555 560Leu Glu Ala Ser Ala Arg Ser Leu Gln Trp Glu Tyr Phe Glu His Val 565 570 575Tyr Gln Thr Met Val Leu Ser Gly Tyr Gln Met Asp Gln Thr Lys His 580 585 590Ala Ser Met Leu Ile Glu Ala Ser Arg Ala Gly Lys Trp Ser Leu Leu 595 600 605Glu His Ala Phe Asp Ala Val Leu Glu Asp Gly Glu Ile Pro His Pro 610 615 620Leu Phe Phe Thr Glu Leu Leu Cys His Ala Thr Ala Lys Gly Asp Phe625 630 635 640Gln Arg Ala Ile Thr Leu Ile Asn Thr Val Ala Leu Ala Ser Phe Gln 645 650 655Ile Ser Glu Glu Glu Trp Thr Asp Leu Phe Glu Glu His Gln Asp Trp 660 665 670Leu Thr Gln Asp Asn Leu His Lys Leu Ser Asp His Leu Ile Glu Cys 675 680 685Asp Tyr Val Ser Glu Pro Thr Val Ser Asn Leu Ser Lys Ser Leu Lys 690 695 700Ser Arg Cys Gly Ser Ser Ser Ser Ser Ala Gln Pro Leu Leu Ala Val705 710 715 720Asp Val Thr Thr Gln Ser Gln Gly Glu Lys Pro Glu Glu Asp Leu Leu 725 730 735Leu Gln Asp Thr Thr Met Glu Asp Asp Asn Ser Ala Asn Gly Glu Ala 740 745 750Trp Glu Phe Thr Glu Thr Glu Leu Glu Thr Leu Gly Leu Glu Glu Leu 755 760 765Glu Ile Asp Asp Asp Glu Glu Ser Ser Asp Ser Asp Ser Leu Ser Val 770 775 780Tyr Asp Ile Leu Lys Glu Trp Glu Glu Ser Ser Lys Lys Glu785 790 7955913PRTArabidopsis thaliana 5Met Ser Leu Ser His Leu Leu Arg Arg Leu Cys Thr Thr Thr Thr Thr1 5 10 15Thr Arg Ser Pro Leu Ser Ile Ser Phe Leu His Gln Arg Ile His Asn 20 25 30Ile Ser Leu Ser Pro Ala Asn Glu Asp Pro Glu Thr Thr Thr Gly Asn 35 40 45Asn Gln Asp Ser Glu Lys Tyr Pro Asn Leu Asn Pro Ile Pro Asn Asp 50 55 60Pro Ser Gln Phe Gln Ile Pro Gln Asn His Thr Pro Pro Ile Pro Tyr65 70 75 80Pro Pro Ile Pro His Arg Thr Met Ala Phe Ser Ser Ala Glu Glu Ala 85 90 95Ala Ala Glu Arg Arg Arg Arg Lys Arg Arg Leu Arg Ile Glu Pro Pro 100 105 110Leu His Ala Leu Arg Arg Asp Pro Ser Ala Pro Pro Pro Lys Arg Asp 115 120 125Pro Asn Ala Pro Arg Leu Pro Asp Ser Thr Ser Ala Leu Val Gly Gln 130 135 140Arg Leu Asn Leu His Asn Arg Val Gln Ser Leu Ile Arg Ala Ser Asp145 150 155 160Leu Asp Ala Ala Ser Lys Leu Ala Arg Gln Ser Val Phe Ser Asn Thr 165 170 175Arg Pro Thr Val Phe Thr Cys Asn Ala Ile Ile Ala Ala Met Tyr Arg 180 185 190Ala Lys Arg Tyr Ser Glu Ser Ile Ser Leu Phe Gln Tyr Phe Phe Lys 195 200 205Gln Ser Asn Ile Val Pro Asn Val Val Ser Tyr Asn Gln Ile Ile Asn 210 215 220Ala His Cys Asp Glu Gly Asn Val Asp Glu Ala Leu Glu Val Tyr Arg225 230 235 240His Ile Leu Ala Asn Ala Pro Phe Ala Pro Ser Ser Val Thr Tyr Arg 245 250 255His Leu Thr Lys Gly Leu Val Gln Ala Gly Arg Ile Gly Asp Ala Ala 260 265 270Ser Leu Leu Arg Glu Met Leu Ser Lys Gly Gln Ala Ala Asp Ser Thr 275 280 285Val Tyr Asn Asn Leu Ile Arg Gly Tyr Leu Asp Leu Gly Asp Phe Asp 290 295 300Lys Ala Val Glu Phe Phe Asp Glu Leu Lys Ser Lys Cys Thr Val Tyr305 310 315 320Asp Gly Ile Val Asn Ala Thr Phe Met Glu Tyr Trp Phe Glu Lys Gly 325 330 335Asn Asp Lys Glu Ala Met Glu Ser Tyr Arg Ser Leu Leu Asp Lys Lys 340 345 350Phe Arg Met His Pro Pro Thr Gly Asn Val Leu Leu Glu Val Phe Leu 355 360 365Lys Phe Gly Lys Lys Asp Glu Ala Trp Ala Leu Phe Asn Glu Met Leu 370 375 380Asp Asn His Ala Pro Pro Asn Ile Leu Ser Val Asn Ser Asp Thr Val385 390 395 400Gly Ile Met Val Asn Glu Cys Phe Lys Met Gly Glu Phe Ser Glu Ala 405 410 415Ile Asn Thr Phe Lys Lys Val Gly Ser Lys Val Thr Ser Lys Pro Phe 420 425 430Val Met Asp Tyr Leu Gly Tyr Cys Asn Ile Val Thr Arg Phe Cys Glu 435 440 445Gln Gly Met Leu Thr Glu Ala Glu Arg Phe Phe Ala Glu Gly Val Ser 450 455 460Arg Ser Leu Pro Ala Asp Ala Pro Ser His Arg Ala Met Ile Asp Ala465 470 475 480Tyr Leu Lys Ala Glu Arg Ile Asp Asp Ala Val Lys Met Leu Asp Arg 485 490 495Met Val Asp Val Asn Leu Arg Val Val Ala Asp Phe Gly Ala Arg Val 500 505 510Phe Gly Glu Leu Ile Lys Asn Gly Lys Leu Thr Glu Ser Ala Glu Val 515 520 525Leu Thr Lys Met Gly Glu Arg Glu Pro Lys Pro Asp Pro Ser Ile Tyr 530 535 540Asp Val Val Val Arg Gly Leu Cys Asp Gly Asp Ala Leu Asp Gln Ala545 550 555 560Lys Asp Ile Val Gly Glu Met Ile Arg His Asn Val Gly Val Thr Thr 565 570 575Val Leu Arg Glu Phe Ile Ile Glu Val Phe Glu Lys Ala Gly Arg Arg 580 585 590Glu Glu Ile Glu Lys Ile Leu Asn Ser Val Ala Arg Pro Val Arg Asn 595 600 605Ala Gly Gln Ser Gly Asn Thr Pro Pro Arg Val Pro Ala Val Phe Gly 610 615 620Thr Thr Pro Ala Ala Pro Gln Gln Pro Arg Asp Arg Ala Pro Trp Thr625 630 635 640Ser Gln Gly Val Val His Ser Asn Ser Gly Trp Ala Asn Gly Thr Ala 645 650 655Gly Gln Thr Ala Gly Gly Ala Tyr Lys Ala Asn Asn Gly Gln Asn Pro 660 665 670Ser Trp Ser Asn Thr Ser Asp Asn Gln Gln Gln Gln Ser Trp Ser Asn 675 680 685Gln Thr Ala Gly Gln Gln Pro Pro Ser Trp Ser Arg Gln Ala Pro Gly 690 695 700Tyr Gln Gln Gln Gln Ser Trp Ser Gln Gln Ser Gly Trp Ser Ser Pro705 710 715 720Ser Gly His Gln Gln Ser Trp Thr Asn Gln Thr Ala Gly Gln Gln Gln 725 730 735Pro Trp Ala Asn Gln Thr Pro Gly Gln Gln Gln Gln Trp Ala Asn Gln 740 745 750Thr Pro Gly Gln Gln Gln Gln Leu Ala Asn Gln Thr Pro Gly Gln Gln 755 760 765Gln Gln Trp Ala Asn Gln Thr Pro Gly Gln Gln Gln Gln Trp Ala Asn 770 775 780Gln Asn Asn Gly His Gln Gln Pro Trp Ala Asn Gln Asn Thr Gly His785 790 795 800Gln Gln Ser Trp Ala Asn Gln Thr Pro Ser Gln Gln Gln Pro Trp Ala 805 810 815Asn Gln Thr Thr Gly Gln Gln Gln Gly Trp Gly Asn Gln Thr Thr Gly 820 825 830Gln Gln Gln Gln Trp Ala Asn Gln Thr Ala Gly Gln Gln Ser Gly Trp 835 840 845Thr Ala Gln Gln Gln Trp Ser Asn Gln Thr Ala Ser His Gln Gln Ser 850 855 860Gln Trp Leu Asn Pro Val Pro Gly Glu Val Ala Asn Gln Thr Pro Trp865 870 875 880Ser Asn Ser Val Asp Ser His Leu Pro Gln Gln Gln Glu Pro Gly Pro 885 890 895Ser His Glu Cys Gln Glu Thr Gln Glu Lys Lys Val Val Glu Leu Arg 900 905 910Asn6196PRTFlabovacterium okeianocoites 6Ala Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His1 5 10 15Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala 20 25 30Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe 35 40 45Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg 50 55 60Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly65 70 75 80Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile 85 90 95Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg 100 105 110Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser 115 120 125Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn 130 135 140Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly145 150 155 160Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys 165 170 175Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly 180 185 190Glu Ile Asn Phe 19575303DNAArtificial Sequencep63-VP64 7cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 60ctacttgttc tttttgcaag atctccacca tggactataa ggaccacgac ggagactaca 120aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga 180aggtcggtat ccccgggggc gaagtgctgt ataggacact gctggccaac tgcgtggctg 240ctgggaacgt gaagaagtcc gaactggtct tcaacaagat gaaggatctg gggttccccc 300tgagcggctt tacctgtgac caaatgctgc tgctgcacaa aaggattgat agaaagaaaa 360tcgctgatgt cctgctgctg atggaaaagg aaaatatcaa gccaagcctg ctgacctaca 420agatcctgat cgatgtgaag ggcgccacca acgacattag cgggatggaa cagattctgg 480aaacaatgaa agacgagggc gtggagctgg atttccaaac acaggccctg acagccaggc 540attactccgg cgctggactg aaagataagg cagaaaaggt gctgaaggaa atggagggag 600agtccctgga agcaaatagg agggccttta aggacctgct gtccatttac gcctccctgg 660gcagagaaga cgaagtgaaa agaatttgga agatttgcga gtccaaacca tactttgagg 720aatccctggc cgctatccaa gcattcggca agctgaataa ggtgcaagaa gccgaggcaa 780tcttcgaaaa gattgtgaag atggatagaa gagcaagctc cagcacatac tccgtcctgc 840tgagagtgta cgtggatcat aagatgctga gcaaaggcaa agacctggtg aagagaatgg 900ccgagagcgg gtgcagaatt gaagccacca cctgggacgc tctgatcaaa ctgtatgtcg 960aggctgggga ggtggaaaaa gccgattccc tgctggataa agccagcaaa caatcccaca 1020ctaaactgat gatgaatagc ttcatgtata tcatggacga gtatagcaag aggggcgacg 1080tgcacaatac cgaaaaaatc tttctgaaaa tgagggaagc cgggtatact agcggatccg 1140gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac gccctcgatg 1200attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc gacatgctcg 1260gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactctagt tgatctagat 1320tctgcagccc tatagtgagt cgtattacgt agatccagac atgataagat acattgatga 1380gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 1440tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 1500cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc 1560ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg 1620cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 1680ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 1740taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 1800cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 1860tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 1920gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 1980atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2040ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2100cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2160tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2220gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2280aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2340tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2400aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2460aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2520ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2580ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2640atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 2700atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 2760tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 2820gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 2880tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 2940gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 3000cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 3060gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 3120atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 3180aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 3240atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 3300aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 3360aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 3420gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 3480gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 3540gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 3600ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 3660ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 3720atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 3780gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 3840tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat

3900agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 3960tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 4020catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 4080aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 4140ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 4200taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca 4260ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga 4320ccatagccaa ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga 4380cctgtgccaa ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga 4440ccccagccaa ttcaatatgg cggacttggc accatgccaa ttcaatatgg cggacttggc 4500actgtgccaa ctggggaggg gtctacttgg cacggtgcca agtttgagga ggggtcttgg 4560ccctgtgcca agtccgccat attgaattgg catggtgcca ataatggcgg ccatattggc 4620tatatgccag gatcaatata taggcaatat ccaatatggc cctatgccaa tatggctatt 4680ggccaggttc aatactatgt attggcccta tgccatatag tattccatat atgggttttc 4740ctattgacgt agatagcccc tcccaatggg cggtcccata taccatatat ggggcttcct 4800aataccgccc atagccactc ccccattgac gtcaatggtc tctatatatg gtctttccta 4860ttgacgtcat atgggcggtc ctattgacgt atatggcgcc tcccccattg acgtcaatta 4920cggtaaatgg cccgcctggc tcaatgccca ttgacgtcaa taggaccacc caccattgac 4980gtcaatggga tggctcattg cccattcata tccgttctca cgccccctat tgacgtcaat 5040gacggtaaat ggcccacttg gcagtacatc aatatctatt aatagtaact tggcaagtac 5100attactattg gaaggacgcc agggtacatt ggcagtactc ccattgacgt caatggcggt 5160aaatggcccg cgatggctgc caagtacatc cccattgacg tcaatgggga ggggcaatga 5220cgcaaatggg cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata 5280taagcaatgc tcgtttaggg aac 530385948DNAArtificial SequencepTac2-VP64 8cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 60ctacttgttc tttttgcaag atctccacca tggactataa ggaccacgac ggagactaca 120aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga 180aggtcggtat ccccgggtcc ctgaacgact ttgcactggt ctttaaggaa ttcgcaggaa 240ggggggattg gcaaagatcc ctgagactgt ttaagtatat gcagaggcaa atctggtgca 300aacccaatga gcatatctat accattatga tttccctgct ggggagagaa ggactgctgg 360ataaatgtct ggaagtgttt gacgaaatgc cttcccaagg agtgagcagg agcgtgttca 420gctacactgc actgattaac gcctacggca gaaacggcag gtacgaaact agcctggagc 480tgctggacag aatgaaaaac gagaagatca gcccaagcat cctgacttat aacacagtga 540tcaatgcttg tgccagaggc ggactggact gggagggcct gctgggcctg ttcgcagaga 600tgaggcacga agggattcaa cccgacatcg tgacttacaa tactctgctg tccgcatgtg 660caattagggg cctgggggac gaagctgaaa tggtcttcag gactatgaat gacggcggaa 720tcgtgcccga tctgaccaca tattcccatc tggtcgagac ctttgggaaa ctgaggagac 780tggagaaggt gtgcgatctg ctgggagaaa tggctagcgg aggctccctg ccagatatta 840cctcctacaa cgtgctgctg gaagcctacg ccaagtccgg ctccatcaag gaggctatgg 900gcgtgtttca tcagatgcaa gccgctggct gtacccccaa tgccaacacc tattccgtcc 960tgctgaatct gttcggccag agcgggagat acgatgacgt gaggcagctg tttctggaaa 1020tgaagagcag caacaccgac cccgacgctg caacatacaa cattctgatc gaggtgtttg 1080gcgagggggg ctacttcaaa gaagtcgtca ccctgttcca cgacatggtg gaggaaaaca 1140tcgagcccga tatggagacc tatgagggga tcatcttcgc ttgcggcaaa ggcggcctgc 1200atgaggacgc taggaagatc ctgcagtaca tgaccgctaa tgacattgtc ccatcctcca 1260aagcttatac cggcgtgatc gaggccttcg gccaggctgc cctgtacgag gaagcactgg 1320tcgcctttaa caccatgcac gaggtcggca gcaacccttc catcgagacc ttccactccc 1380tgctgtatag cttcgccaga ggcgggctgg tgaaggagtc cgaggcaatc ctgagcaggc 1440tggtcgattc cggcatcccc aggaacagag acacctttaa tgctcaaatt gaggcctaca 1500aacagggggg gaagttcgaa gaggctgtga agacctacgt cgacatggaa aagagcaggt 1560gcgaccccga cgagaggacc ctggaggccg tcctgtccgt gtattccttc gcaagactgg 1620tggatgagtg cagggaacag tttgaagaaa tgaaggccag cgacattctg cccagcatta 1680tgtgctactg catgatgctg gcagtgtacg ggaagaccga gaggtgggac gacgtgaacg 1740aactgctgga ggagatgctg agcaacaggg tcagcaacgg atccggacgg gctgacgcat 1800tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca 1860tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg 1920atgatttcga cctggacatg ctgattaact ctagttgatc tagattctgc agccctatag 1980tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac 2040aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 2100tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 2160tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc caatgcattg 2220ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca 2280tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 2340gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 2400gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 2460atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 2520actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2580gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2640cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2700ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2760ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2820ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 2880agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2940cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 3000aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 3060gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 3120agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3180ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3240cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3300tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3360aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3420tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3480atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3540cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3600gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3660gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3720tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 3780tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3840tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3900aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3960atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 4020tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 4080catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 4140aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 4200tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4260gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4320tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4380tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg 4440taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 4500accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 4560tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 4620aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 4680gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 4740ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 4800gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 4860ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt 4920gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccata gccaattcaa 4980tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt gccaattcaa 5040tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca gccaattcaa 5100tatggcggac ttggcaccat gccaattcaa tatggcggac ttggcactgt gccaactggg 5160gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg tgccaagtcc 5220gccatattga attggcatgg tgccaataat ggcggccata ttggctatat gccaggatca 5280atatataggc aatatccaat atggccctat gccaatatgg ctattggcca ggttcaatac 5340tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt gacgtagata 5400gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac cgcccatagc 5460cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac gtcatatggg 5520cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta aatggcccgc 5580ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa tgggatggct 5640cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg taaatggccc 5700acttggcagt acatcaatat ctattaatag taacttggca agtacattac tattggaagg 5760acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg 5820gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc 5880cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc aatgctcgtt 5940tagggaac 594895531DNAArtificial SequenceGUN1-VP64 9cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 60ctacttgttc tttttgcaag atctccacca tggactataa ggaccacgac ggagactaca 120aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga 180aggtcggtat ccccgggcaa ggcaagctgg caagcgccat gatctccacc ctgggcaggt 240acggaaaggt gaccattgcc aagaggatct tcgagaccgc cttcgcaggc gggtacggca 300acaccgtgta tgctttttcc gccctgatta gcgcatatgg cagaagcggc ctgcacgaag 360aggccattag cgtgtttaac agcatgaagg agtatggact gaggcccaac ctggtgacct 420acaacgccgt cattgatgct tgcggcaagg gcggcatgga attcaagcag gtggccaagt 480tcttcgatga aatgcagagg aacggcgtgc agcctgacag aattacattc aatagcctgc 540tggctgtgtg cagcagaggg ggcctgtggg aggcagctag gaatctgttt gacgagatga 600ccaatagaag gatcgagcag gacgtgttct cctataatac actgctggac gccatttgta 660aaggcgggca aatggacctg gccttcgaaa tcctggccca gatgcccgtc aaaaggatca 720tgcccaacgt ggtcagctac tccacagtca tcgacgggtt cgccaaggct ggcaggtttg 780atgaagcact gaacctgttc ggggaaatga gatacctggg aatcgccctg gacagggtga 840gctacaacac cctgctgagc atctacacta aggtcggcag atccgaggaa gccctggaca 900tcctgaggga aatggcctcc gtgggcatta agaaggacgt cgtgacatac aatgccctgc 960tgggcggcta cggcaaacag ggcaagtacg acgaggtcaa gaaggtcttc acagagatga 1020agagggaaca cgtgctgcca aatctgctga cttattccac tctgattgat ggctactcca 1080aaggcggact gtacaaggaa gccatggaga ttttcagaga gttcaagagc gctggcctga 1140gagccgatgt cgtgctgtat tccgcactga tcgatgcact gtgcaaaaac ggcctggtcg 1200gcagcgccgt gagcctgatc gacgagatga ccaaggaggg aattagcccc aatgtggtga 1260cttacaatag catcattgat gctttcggca gaagcgccac catggacaga tccgccgact 1320atagcaacgg cggcagcctg ccattttcct ccagcgccct gggatccgga cgggctgacg 1380cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat tttgaccttg 1440acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc agtgacgccc 1500ttgatgattt cgacctggac atgctgatta actctagttg atctagattc tgcagcccta 1560tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac 1620cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 1680atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 1740gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca 1800ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 1860tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 1920cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1980attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 2040tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 2100ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 2160gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2220ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2280cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2340ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2400accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2460catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2520gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2580tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 2640agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2700actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2760gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 2820aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 2880gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 2940aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3000atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3060gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3120atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3180ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3240cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3300agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3360cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3420tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3480agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3540gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 3600gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 3660ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 3720tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 3780tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3840gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 3900caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 3960atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa 4020ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 4080ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 4140ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 4200tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 4260caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 4320gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 4380aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 4440ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact 4500gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc atagccaatt 4560caatatggcg tatatggact catgccaatt caatatggtg gatctggacc tgtgccaatt 4620caatatggcg tatatggact cgtgccaatt caatatggtg gatctggacc ccagccaatt 4680caatatggcg gacttggcac catgccaatt caatatggcg gacttggcac tgtgccaact 4740ggggaggggt ctacttggca cggtgccaag tttgaggagg ggtcttggcc ctgtgccaag 4800tccgccatat tgaattggca tggtgccaat aatggcggcc atattggcta tatgccagga 4860tcaatatata ggcaatatcc aatatggccc tatgccaata tggctattgg ccaggttcaa 4920tactatgtat tggccctatg ccatatagta ttccatatat gggttttcct attgacgtag 4980atagcccctc ccaatgggcg gtcccatata ccatatatgg ggcttcctaa taccgcccat 5040agccactccc ccattgacgt caatggtctc tatatatggt ctttcctatt gacgtcatat 5100gggcggtcct attgacgtat atggcgcctc ccccattgac gtcaattacg gtaaatggcc 5160cgcctggctc aatgcccatt gacgtcaata ggaccaccca ccattgacgt caatgggatg 5220gctcattgcc cattcatatc cgttctcacg ccccctattg acgtcaatga cggtaaatgg 5280cccacttggc agtacatcaa tatctattaa tagtaacttg gcaagtacat tactattgga 5340aggacgccag ggtacattgg cagtactccc attgacgtca atggcggtaa atggcccgcg 5400atggctgcca agtacatccc cattgacgtc aatggggagg ggcaatgacg caaatgggcg 5460ttccattgac gtaaatgggc ggtaggcgtg cctaatggga ggtctatata agcaatgctc 5520gtttagggaa c 5531105135DNAArtificial SequencepminCMV-luc2 10ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag cttactagtg 60tcgaggtagg cgtgtacggt gggaggccta tataagcaga gctcgtttag tgaaccgtca 120gatcgcctgg aggtaccgcc accatggaag atgccaaaaa cattaagaag ggcccagcgc 180cattctaccc actcgaagac gggaccgccg gcgagcagct gcacaaagcc atgaagcgct 240acgccctggt gcccggcacc atcgccttta ccgacgcaca tatcgaggtg gacattacct 300acgccgagta cttcgagatg agcgttcggc tggcagaagc tatgaagcgc tatgggctga 360atacaaacca tcggatcgtg gtgtgcagcg agaatagctt gcagttcttc atgcccgtgt 420tgggtgccct gttcatcggt gtggctgtgg ccccagctaa cgacatctac aacgagcgcg 480agctgctgaa cagcatgggc atcagccagc ccaccgtcgt attcgtgagc aagaaagggc 540tgcaaaagat cctcaacgtg caaaagaagc taccgatcat acaaaagatc atcatcatgg 600atagcaagac cgactaccag ggcttccaaa gcatgtacac cttcgtgact tcccatttgc 660cacccggctt caacgagtac gacttcgtgc ccgagagctt cgaccgggac aaaaccatcg 720ccctgatcat gaacagtagt ggcagtaccg gattgcccaa gggcgtagcc ctaccgcacc 780gcaccgcttg tgtccgattc agtcatgccc gcgaccccat cttcggcaac cagatcatcc 840ccgacaccgc tatcctcagc gtggtgccat ttcaccacgg cttcggcatg ttcaccacgc 900tgggctactt gatctgcggc tttcgggtcg tgctcatgta ccgcttcgag gaggagctat 960tcttgcgcag cttgcaagac tataagattc aatctgccct gctggtgccc acactattta 1020gcttcttcgc taagagcact ctcatcgaca agtacgacct aagcaacttg cacgagatcg 1080ccagcggcgg ggcgccgctc agcaaggagg taggtgaggc cgtggccaaa cgcttccacc 1140taccaggcat ccgccagggc tacggcctga cagaaacaac cagcgccatt ctgatcaccc 1200ccgaagggga cgacaagcct ggcgcagtag gcaaggtggt gcccttcttc gaggctaagg 1260tggtggactt ggacaccggt aagacactgg gtgtgaacca gcgcggcgag ctgtgcgtcc 1320gtggccccat gatcatgagc ggctacgtta acaaccccga ggctacaaac gctctcatcg 1380acaaggacgg ctggctgcac agcggcgaca tcgcctactg ggacgaggac gagcacttct 1440tcatcgtgga ccggctgaag agcctgatca aatacaaggg ctaccaggta gccccagccg 1500aactggagag catcctgctg caacacccca acatcttcga cgccggggtc gccggcctgc 1560ccgacgacga tgccggcgag ctgcccgccg cagtcgtcgt gctggaacac ggtaaaacca 1620tgaccgagaa ggagatcgtg gactatgtgg ccagccaggt tacaaccgcc aagaagctgc 1680gcggtggtgt tgtgttcgtg gacgaggtgc ctaaaggact gaccggcaag ttggacgccc 1740gcaagatccg cgagattctc attaaggcca agaagggcgg caagatcgcc gtgaattctt 1800aactgcagtt aatctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca 1860ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 1920tttgtgatgc

tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 1980acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 2040agtaaaacct ctacaaatgt ggtaaaatcg ataaggatct gaacgatgga gcggagaatg 2100ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc 2160tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc 2220acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc 2280tggggacttt ccacacccta actgacacac attccacagc ggatccgtcg accgatgccc 2340ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc 2400gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc 2460cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 2520tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 2580gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 2640ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 2700aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 2760tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 2820ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 2880gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 2940tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 3000caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 3060ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 3120cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 3180ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 3240cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 3300gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 3360aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 3420acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 3480gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 3540cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 3600cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 3660tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 3720cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 3780gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 3840cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 3900ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 3960gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 4020taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 4080gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 4140acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 4200aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 4260cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 4320atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 4380gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 4440cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 4500tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4560ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 4620tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4680taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 4740tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4800aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttgccatt cgccattcag 4860gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagcccaa 4920gctaccatga taagtaagta atattaaggt acgggaggta cttggagcgg ccgcaataaa 4980atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca 5040tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt 5100gcaagtgcag gtgccagaac atttctctat cgata 5135119DNAArtificial SequenceTarget sequence in p63 11tctatcact 91215DNAArtificial SequenceTarget sequence in pTac2 12aactttcgtc actca 151311DNAArtificial SequenceTarget sequence in GUN1 13aatttgtcga t 11145204DNAArtificial Sequencep63-4x target 14ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag ttctatcact 60ttgtttcttc tatcacttga attcttctat cacttatctt cttctatcac ttcagttcgc 120ttactagtgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt 180gaaccgtcag atcgcctgga ggtaccgcca ccatggaaga tgccaaaaac attaagaagg 240gcccagcgcc attctaccca ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca 300tgaagcgcta cgccctggtg cccggcacca tcgcctttac cgacgcacat atcgaggtgg 360acattaccta cgccgagtac ttcgagatga gcgttcggct ggcagaagct atgaagcgct 420atgggctgaa tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg cagttcttca 480tgcccgtgtt gggtgccctg ttcatcggtg tggctgtggc cccagctaac gacatctaca 540acgagcgcga gctgctgaac agcatgggca tcagccagcc caccgtcgta ttcgtgagca 600agaaagggct gcaaaagatc ctcaacgtgc aaaagaagct accgatcata caaaagatca 660tcatcatgga tagcaagacc gactaccagg gcttccaaag catgtacacc ttcgtgactt 720cccatttgcc acccggcttc aacgagtacg acttcgtgcc cgagagcttc gaccgggaca 780aaaccatcgc cctgatcatg aacagtagtg gcagtaccgg attgcccaag ggcgtagccc 840taccgcaccg caccgcttgt gtccgattca gtcatgcccg cgaccccatc ttcggcaacc 900agatcatccc cgacaccgct atcctcagcg tggtgccatt tcaccacggc ttcggcatgt 960tcaccacgct gggctacttg atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg 1020aggagctatt cttgcgcagc ttgcaagact ataagattca atctgccctg ctggtgccca 1080cactatttag cttcttcgct aagagcactc tcatcgacaa gtacgaccta agcaacttgc 1140acgagatcgc cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac 1200gcttccacct accaggcatc cgccagggct acggcctgac agaaacaacc agcgccattc 1260tgatcacccc cgaaggggac gacaagcctg gcgcagtagg caaggtggtg cccttcttcg 1320aggctaaggt ggtggacttg gacaccggta agacactggg tgtgaaccag cgcggcgagc 1380tgtgcgtccg tggccccatg atcatgagcg gctacgttaa caaccccgag gctacaaacg 1440ctctcatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg gacgaggacg 1500agcacttctt catcgtggac cggctgaaga gcctgatcaa atacaagggc taccaggtag 1560ccccagccga actggagagc atcctgctgc aacaccccaa catcttcgac gccggggtcg 1620ccggcctgcc cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg 1680gtaaaaccat gaccgagaag gagatcgtgg actatgtggc cagccaggtt acaaccgcca 1740agaagctgcg cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg accggcaagt 1800tggacgcccg caagatccgc gagattctca ttaaggccaa gaagggcggc aagatcgccg 1860tgaattctta actgcagtta atctagagtc ggggcggccg gccgcttcga gcagacatga 1920taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 1980tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 2040ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 2100tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctg aacgatggag 2160cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac 2220tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg 2280ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc 2340tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagcg gatccgtcga 2400ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact 2460atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca 2520gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 2580ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 2640aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 2700ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 2760gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 2820cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 2880gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 2940tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 3000cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 3060cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 3120gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 3180agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 3240cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 3300tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 3360tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 3420ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 3480cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 3540cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 3600accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 3660ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 3720ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 3780tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 3840acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 3900tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 3960actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 4020ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 4080aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 4140ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 4200cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 4260aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 4320actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 4380cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 4440ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 4500tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 4560cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 4620tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 4680tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 4740cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 4800ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 4860gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc 4920gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 4980ccagcccaag ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc 5040cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata 5100gtactaacat acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct 5160gtccccagtg caagtgcagg tgccagaaca tttctctatc gata 5204155272DNAArtificial Sequencep63-8x target 15ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag ttctatcact 60tccatggttc tatcacttca cgacttctat cactttgttt cttctatcac ttgaattctt 120ctatcactta agttcttcta tcacttttcg aattctatca cttatcttct tctatcactt 180cagttcgctt actagtgtcg aggtaggcgt gtacggtggg aggcctatat aagcagagct 240cgtttagtga accgtcagat cgcctggagg taccgccacc atggaagatg ccaaaaacat 300taagaagggc ccagcgccat tctacccact cgaagacggg accgccggcg agcagctgca 360caaagccatg aagcgctacg ccctggtgcc cggcaccatc gcctttaccg acgcacatat 420cgaggtggac attacctacg ccgagtactt cgagatgagc gttcggctgg cagaagctat 480gaagcgctat gggctgaata caaaccatcg gatcgtggtg tgcagcgaga atagcttgca 540gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg gctgtggccc cagctaacga 600catctacaac gagcgcgagc tgctgaacag catgggcatc agccagccca ccgtcgtatt 660cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa aagaagctac cgatcataca 720aaagatcatc atcatggata gcaagaccga ctaccagggc ttccaaagca tgtacacctt 780cgtgacttcc catttgccac ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga 840ccgggacaaa accatcgccc tgatcatgaa cagtagtggc agtaccggat tgcccaaggg 900cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt catgcccgcg accccatctt 960cggcaaccag atcatccccg acaccgctat cctcagcgtg gtgccatttc accacggctt 1020cggcatgttc accacgctgg gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg 1080cttcgaggag gagctattct tgcgcagctt gcaagactat aagattcaat ctgccctgct 1140ggtgcccaca ctatttagct tcttcgctaa gagcactctc atcgacaagt acgacctaag 1200caacttgcac gagatcgcca gcggcggggc gccgctcagc aaggaggtag gtgaggccgt 1260ggccaaacgc ttccacctac caggcatccg ccagggctac ggcctgacag aaacaaccag 1320cgccattctg atcacccccg aaggggacga caagcctggc gcagtaggca aggtggtgcc 1380cttcttcgag gctaaggtgg tggacttgga caccggtaag acactgggtg tgaaccagcg 1440cggcgagctg tgcgtccgtg gccccatgat catgagcggc tacgttaaca accccgaggc 1500tacaaacgct ctcatcgaca aggacggctg gctgcacagc ggcgacatcg cctactggga 1560cgaggacgag cacttcttca tcgtggaccg gctgaagagc ctgatcaaat acaagggcta 1620ccaggtagcc ccagccgaac tggagagcat cctgctgcaa caccccaaca tcttcgacgc 1680cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct 1740ggaacacggt aaaaccatga ccgagaagga gatcgtggac tatgtggcca gccaggttac 1800aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac gaggtgccta aaggactgac 1860cggcaagttg gacgcccgca agatccgcga gattctcatt aaggccaaga agggcggcaa 1920gatcgccgtg aattcttaac tgcagttaat ctagagtcgg ggcggccggc cgcttcgagc 1980agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2040atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2100taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2160ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatctgaa 2220cgatggagcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg 2280ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 2340gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt 2400ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagcgga 2460tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg 2520gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg 2580tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 2640cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 2700aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2760gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2820tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 2880agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 2940ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 3000taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 3060gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 3120gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 3180ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 3240ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 3300gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 3360caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 3420taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 3480aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 3540tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 3600tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 3660gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 3720gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 3780aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 3840gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3900ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 3960tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 4020atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 4080ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 4140ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 4200ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 4260atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 4320gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 4380tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 4440ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 4500acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt 4560gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4620gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4680gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4740tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4800ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4860atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4920aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 4980tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 5040ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt 5100ggagcggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg 5160aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa 5220aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga ta 5272165228DNAArtificial SequencepTac2-4x target 16ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag taactttcgt 60cactcattgt ttctaacttt cgtcactcat ggattctaac tttcgtcact catatcttct 120aactttcgtc actcatcagt tcgcttacta gtgtcgaggt aggcgtgtac ggtgggaggc 180ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggaggtacc gccaccatgg 240aagatgccaa aaacattaag aagggcccag cgccattcta cccactcgaa gacgggaccg 300ccggcgagca gctgcacaaa gccatgaagc gctacgccct ggtgcccggc accatcgcct 360ttaccgacgc acatatcgag gtggacatta cctacgccga gtacttcgag atgagcgttc 420ggctggcaga agctatgaag cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca 480gcgagaatag cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg 540tggccccagc taacgacatc tacaacgagc gcgagctgct gaacagcatg ggcatcagcc 600agcccaccgt cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac gtgcaaaaga 660agctaccgat catacaaaag atcatcatca tggatagcaa gaccgactac cagggcttcc 720aaagcatgta caccttcgtg acttcccatt tgccacccgg cttcaacgag tacgacttcg 780tgcccgagag cttcgaccgg gacaaaacca tcgccctgat catgaacagt agtggcagta 840ccggattgcc caagggcgta gccctaccgc accgcaccgc

ttgtgtccga ttcagtcatg 900cccgcgaccc catcttcggc aaccagatca tccccgacac cgctatcctc agcgtggtgc 960catttcacca cggcttcggc atgttcacca cgctgggcta cttgatctgc ggctttcggg 1020tcgtgctcat gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa gactataaga 1080ttcaatctgc cctgctggtg cccacactat ttagcttctt cgctaagagc actctcatcg 1140acaagtacga cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg ctcagcaagg 1200aggtaggtga ggccgtggcc aaacgcttcc acctaccagg catccgccag ggctacggcc 1260tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag cctggcgcag 1320taggcaaggt ggtgcccttc ttcgaggcta aggtggtgga cttggacacc ggtaagacac 1380tgggtgtgaa ccagcgcggc gagctgtgcg tccgtggccc catgatcatg agcggctacg 1440ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg cacagcggcg 1500acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga 1560tcaaatacaa gggctaccag gtagccccag ccgaactgga gagcatcctg ctgcaacacc 1620ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc gagctgcccg 1680ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc gtggactatg 1740tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc gtggacgagg 1800tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt ctcattaagg 1860ccaagaaggg cggcaagatc gccgtgaatt cttaactgca gttaatctag agtcggggcg 1920gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 1980gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 2040ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 2100ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa 2160tcgataagga tctgaacgat ggagcggaga atgggcggaa ctgggcggag ttaggggcgg 2220gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat 2280acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg 2340catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca 2400cacattccac agcggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc 2460ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg 2520caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg 2580ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 2640cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 2700gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 2760tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 2820ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 2880atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 2940gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3000tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3060cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3120cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 3180tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3240cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3300cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3360gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 3420gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 3480gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 3540ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 3600atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 3660agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 3720ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 3780tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 3840ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 3900caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 3960gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 4020atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 4080accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 4140aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4200gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 4260tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 4320aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 4380ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 4440aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc 4500attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 4560agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 4620tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 4680ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 4740ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 4800aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 4860ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 4920attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 4980ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa 5040ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt 5100gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa 5160caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc 5220tatcgata 5228175320DNAArtificial SequencepTac2-8x target 17ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag taactttcgt 60cactcatcca tggtaacttt cgtcactcat cacgactaac tttcgtcact cattgtttct 120aactttcgtc actcatgaat tctaactttc gtcactcata agttctaact ttcgtcactc 180atttcgaata actttcgtca ctcatatctt ctaactttcg tcactcatca gttcgcttac 240tagtgtcgag gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac 300cgtcagatcg cctggaggta ccgccaccat ggaagatgcc aaaaacatta agaagggccc 360agcgccattc tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa 420gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat 480tacctacgcc gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg 540gctgaataca aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc 600cgtgttgggt gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga 660gcgcgagctg ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa 720agggctgcaa aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat 780catggatagc aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca 840tttgccaccc ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac 900catcgccctg atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc 960gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat 1020catccccgac accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac 1080cacgctgggc tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga 1140gctattcttg cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact 1200atttagcttc ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga 1260gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt 1320ccacctacca ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat 1380cacccccgaa ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc 1440taaggtggtg gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg 1500cgtccgtggc cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct 1560catcgacaag gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca 1620cttcttcatc gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc 1680agccgaactg gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg 1740cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa 1800aaccatgacc gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa 1860gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga 1920cgcccgcaag atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgaa 1980ttcttaactg cagttaatct agagtcgggg cggccggccg cttcgagcag acatgataag 2040atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 2100tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 2160caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta 2220aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatctgaacg atggagcgga 2280gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg 2340gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac 2400tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 2460gagcctgggg actttccaca ccctaactga cacacattcc acagcggatc cgtcgaccga 2520tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg 2580tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc 2640tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 2700tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 2760aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 2820tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2880tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2940cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3000agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 3060tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 3120aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3180ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3240cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 3300accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 3360ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 3420ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 3480gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 3540aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 3600gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 3660gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3720cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 3780gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 3840gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 3900ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 3960tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 4020ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 4080cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 4140accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 4200cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 4260tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 4320cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4380acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4440atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4500tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 4560aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 4620cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 4680tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 4740gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 4800tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 4860ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 4920tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 4980taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg ccattcgcca 5040ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 5100cccaagctac catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca 5160ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac 5220taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc 5280ccagtgcaag tgcaggtgcc agaacatttc tctatcgata 5320185212DNAArtificial SequenceGUN1-4x target 18ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag taatttgtcg 60atttgtttct aatttgtcga ttgaattcta atttgtcgat tatcttctaa tttgtcgatt 120cagttcgctt actagtgtcg aggtaggcgt gtacggtggg aggcctatat aagcagagct 180cgtttagtga accgtcagat cgcctggagg taccgccacc atggaagatg ccaaaaacat 240taagaagggc ccagcgccat tctacccact cgaagacggg accgccggcg agcagctgca 300caaagccatg aagcgctacg ccctggtgcc cggcaccatc gcctttaccg acgcacatat 360cgaggtggac attacctacg ccgagtactt cgagatgagc gttcggctgg cagaagctat 420gaagcgctat gggctgaata caaaccatcg gatcgtggtg tgcagcgaga atagcttgca 480gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg gctgtggccc cagctaacga 540catctacaac gagcgcgagc tgctgaacag catgggcatc agccagccca ccgtcgtatt 600cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa aagaagctac cgatcataca 660aaagatcatc atcatggata gcaagaccga ctaccagggc ttccaaagca tgtacacctt 720cgtgacttcc catttgccac ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga 780ccgggacaaa accatcgccc tgatcatgaa cagtagtggc agtaccggat tgcccaaggg 840cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt catgcccgcg accccatctt 900cggcaaccag atcatccccg acaccgctat cctcagcgtg gtgccatttc accacggctt 960cggcatgttc accacgctgg gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg 1020cttcgaggag gagctattct tgcgcagctt gcaagactat aagattcaat ctgccctgct 1080ggtgcccaca ctatttagct tcttcgctaa gagcactctc atcgacaagt acgacctaag 1140caacttgcac gagatcgcca gcggcggggc gccgctcagc aaggaggtag gtgaggccgt 1200ggccaaacgc ttccacctac caggcatccg ccagggctac ggcctgacag aaacaaccag 1260cgccattctg atcacccccg aaggggacga caagcctggc gcagtaggca aggtggtgcc 1320cttcttcgag gctaaggtgg tggacttgga caccggtaag acactgggtg tgaaccagcg 1380cggcgagctg tgcgtccgtg gccccatgat catgagcggc tacgttaaca accccgaggc 1440tacaaacgct ctcatcgaca aggacggctg gctgcacagc ggcgacatcg cctactggga 1500cgaggacgag cacttcttca tcgtggaccg gctgaagagc ctgatcaaat acaagggcta 1560ccaggtagcc ccagccgaac tggagagcat cctgctgcaa caccccaaca tcttcgacgc 1620cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct 1680ggaacacggt aaaaccatga ccgagaagga gatcgtggac tatgtggcca gccaggttac 1740aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac gaggtgccta aaggactgac 1800cggcaagttg gacgcccgca agatccgcga gattctcatt aaggccaaga agggcggcaa 1860gatcgccgtg aattcttaac tgcagttaat ctagagtcgg ggcggccggc cgcttcgagc 1920agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 1980atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2040taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2100ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatctgaa 2160cgatggagcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg 2220ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg 2280gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt 2340ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagcgga 2400tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg 2460gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg 2520tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 2580cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 2640aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2700gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2760tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 2820agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 2880ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 2940taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 3000gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 3060gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 3120ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 3180ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 3240gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 3300caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 3360taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 3420aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 3480tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 3540tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 3600gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 3660gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 3720aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 3780gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3840ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 3900tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 3960atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 4020ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 4080ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 4140ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 4200atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 4260gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 4320tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 4380ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 4440acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt 4500gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4560gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4620gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4680tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4740ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4800atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4860aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 4920tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 4980ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt 5040ggagcggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg 5100aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa 5160aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga ta 5212195288DNAArtificial

SequenceGUN1-8x target 19ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgatatcaag taatttgtcg 60attccatggt aatttgtcga ttcacgacta atttgtcgat ttgtttctaa tttgtcgatt 120gaattctaat ttgtcgatta agttctaatt tgtcgatttt cgaataattt gtcgattatc 180ttctaatttg tcgattcagt tcgcttacta gtgtcgaggt aggcgtgtac ggtgggaggc 240ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggaggtacc gccaccatgg 300aagatgccaa aaacattaag aagggcccag cgccattcta cccactcgaa gacgggaccg 360ccggcgagca gctgcacaaa gccatgaagc gctacgccct ggtgcccggc accatcgcct 420ttaccgacgc acatatcgag gtggacatta cctacgccga gtacttcgag atgagcgttc 480ggctggcaga agctatgaag cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca 540gcgagaatag cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg 600tggccccagc taacgacatc tacaacgagc gcgagctgct gaacagcatg ggcatcagcc 660agcccaccgt cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac gtgcaaaaga 720agctaccgat catacaaaag atcatcatca tggatagcaa gaccgactac cagggcttcc 780aaagcatgta caccttcgtg acttcccatt tgccacccgg cttcaacgag tacgacttcg 840tgcccgagag cttcgaccgg gacaaaacca tcgccctgat catgaacagt agtggcagta 900ccggattgcc caagggcgta gccctaccgc accgcaccgc ttgtgtccga ttcagtcatg 960cccgcgaccc catcttcggc aaccagatca tccccgacac cgctatcctc agcgtggtgc 1020catttcacca cggcttcggc atgttcacca cgctgggcta cttgatctgc ggctttcggg 1080tcgtgctcat gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa gactataaga 1140ttcaatctgc cctgctggtg cccacactat ttagcttctt cgctaagagc actctcatcg 1200acaagtacga cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg ctcagcaagg 1260aggtaggtga ggccgtggcc aaacgcttcc acctaccagg catccgccag ggctacggcc 1320tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag cctggcgcag 1380taggcaaggt ggtgcccttc ttcgaggcta aggtggtgga cttggacacc ggtaagacac 1440tgggtgtgaa ccagcgcggc gagctgtgcg tccgtggccc catgatcatg agcggctacg 1500ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg cacagcggcg 1560acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga 1620tcaaatacaa gggctaccag gtagccccag ccgaactgga gagcatcctg ctgcaacacc 1680ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc gagctgcccg 1740ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc gtggactatg 1800tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc gtggacgagg 1860tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt ctcattaagg 1920ccaagaaggg cggcaagatc gccgtgaatt cttaactgca gttaatctag agtcggggcg 1980gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 2040gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 2100ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 2160ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa 2220tcgataagga tctgaacgat ggagcggaga atgggcggaa ctgggcggag ttaggggcgg 2280gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat 2340acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg 2400catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca 2460cacattccac agcggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc 2520ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg 2580caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg 2640ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 2700cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 2760gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 2820tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 2880ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 2940atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 3000gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3060tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3120cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3180cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 3240tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3300cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3360cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3420gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 3480gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 3540gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 3600ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 3660atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 3720agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 3780ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 3840tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 3900ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 3960caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 4020gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 4080atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 4140accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 4200aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4260gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 4320tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 4380aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 4440ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 4500aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc 4560attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 4620agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 4680tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 4740ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 4800ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 4860aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 4920ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 4980attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 5040ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa 5100ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt 5160gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa 5220caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc 5280tatcgata 52882035PRTArabidopsis thaliana 20Val Thr Tyr Asn Thr Leu Ile Ser Gly Leu Cys Lys Ala Gly Arg Leu1 5 10 15Glu Glu Ala Leu Glu Leu Phe Glu Glu Met Lys Glu Lys Gly Ile Ala 20 25 30Pro Asp Val 352131PRTArabidopsis thaliana 21Val Val Tyr Asn Ala Leu Ile Asp Met Tyr Ala Lys Cys Gly Asp Leu1 5 10 15Glu Glu Ala Arg Lys Val Phe Asp Glu Met Pro Glu Arg Asp Val 20 25 302235PRTArabidopsis thaliana 22Phe Thr Leu Ala Ser Val Leu Lys Ala Cys Ala Ser Leu Gly Ala Leu1 5 10 15Ser Leu Gly Lys Gln Ile His Gly Tyr Val Ile Lys Ser Gly Phe Asp 20 25 30Ser Asp Glu 352336PRTArabidopsis thaliana 23Val Thr Phe Leu Gly Val Leu Ser Ala Cys Ser His Ser Gly Leu Val1 5 10 15Glu Glu Gly Leu Glu Tyr Phe Glu Ser Met Lys Glu Lys Tyr Gly Ile 20 25 30Glu Pro Asp Glu 35

Claims

1. A method for modifying a genetic substance of a cell, the method comprising: designing a DNA binding protein; determining a DNA base sequence coding for an amino acid sequence of a fused protein comprising a functional region and a DNA binding region consisting of a DNA-binding protein; cloning said DNA base sequence; preparing a vector carrying said DNA base sequence and a cell containing a DNA having at least 9 target DNA bases as a target DNA base sequence; and introducing the vector into the cell so that the DNA binding region of the fused protein binds to the DNA having the at least 9 target DNA bases, and therefore the functional region modifies the DNA having the at least 9 target DNA bases, wherein the DNA-binding protein contains at least 9 PPR motifs for binding to the at least 9 target DNA bases, respectively, and having a structure of the following formula 1: (Helix A)-X-(Helix B)-L (Formula 1) (wherein, in the formula 1: Helix A is a part that can form an .alpha.-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an .alpha.-helix structure; and L is a part consisting of 2 to 7 amino acids), wherein, position numbers of amino acids in the PPR motifs are the same as PF01535 in Pfam under the following definitions: the first amino acid of Helix A is referred to as Number 1 amino acid (Number 1 AA), the fourth amino acid as Number 4 amino acid (Number 4 AA), and when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (when there is no amino acid insertion between the PPR motifs), the -2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n); when a non-PPR motif consisting of 1 to 20 amino acids exists between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the -2nd amino acid; or when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M.sub.n) is referred to as Number "ii" (-2) amino acid (Number "ii" (-2) AA), each PPR motif (M.sub.n) contained in the DNA-binding protein is a PPR motif having a specific combination of amino acids as the three amino acids of Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, such that the DNA-binding protein has at least 9 combinations of the three amino acids corresponding to the respective target DNA bases of the target DNA base sequence, and each combination of the three amino acids is determined according to any one of the following definitions: (2-1) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA are an arbitrary amino acid, glycine, and aspartic acid, respectively; (2-2) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are glutamic acid, glycine, and aspartic acid, respectively; (2-3) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, glycine, and asparagine, respectively; (2-4) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are glutamic acid, glycine, and asparagine, respectively; (2-5) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, glycine, and serine, respectively; (2-7) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, isoleucine, and asparagine, respectively; (2-12) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, methionine, and aspartic acid, respectively; (2-13) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, methionine, and aspartic acid, respectively; (2-15) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, asparagine, and aspartic acid, respectively; (2-16) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are phenylalanine, asparagine, and aspartic acid, respectively; (2-17) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are glycine, asparagine, and aspartic acid, respectively; (2-18) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, asparagine, and aspartic acid, respectively; (2-19) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are threonine, asparagine, and aspartic acid, respectively; (2-20) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA are valine, asparagine, and aspartic acid, respectively; (2-21) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA are tyrosine, asparagine, and aspartic acid, respectively; (2-22) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, asparagine, and asparagine, respectively; (2-23) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, asparagine, and asparagine, respectively; (2-24) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are serine, asparagine, and asparagine, respectively; (2-25) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, asparagine, and asparagine, respectively; (2-26) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, asparagine, and serine, respectively; (2-27) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, asparagine, and serine, respectively; (2-28) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, asparagine, and threonine, respectively; (2-29) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, asparagine, and threonine, respectively; (2-30) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, asparagine, and tryptophan, respectively; (2-31) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, asparagine, and tryptophan, respectively; (2-33) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, proline, and aspartic acid, respectively; (2-34) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are phenylalanine, proline, and aspartic acid, respectively; (2-35) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are tyrosine, proline, and aspartic acid, respectively; (2-37) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, serine, and asparagine, respectively; (2-38) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are phenylalanine, serine, and asparagine, respectively; (2-39) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, serine, and asparagine, respectively; (2-41) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, threonine, and aspartic acid, respectively; (2-42) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, threonine, and aspartic acid, respectively; (2-43) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, threonine, and asparagine, respectively; (2-44) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are phenylalanine, threonine, and asparagine, respectively; (2-45) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, threonine, and asparagine, respectively; (2-46) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are valine, threonine, and asparagine, respectively; (2-48) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are isoleucine, valine, and aspartic acid, respectively; (2-49) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, valine, and glycine, respectively; and (2-50) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number "ii" (-2) AA, are an arbitrary amino acid, valine, and threonine, respectively.

2. The method according to claim 1, wherein the functional region is fused to the DNA-binding protein on the C-terminus side of the protein.

3. The method according to claim 1, wherein the functional region is a DNA-cleaving enzyme, or a nuclease domain thereof, or a transcription control domain, and the complex functions as a target sequence-specific DNA-cleaving enzyme or transcription control factor.

4. The method according to claim 3, wherein the DNA-cleaving enzyme is the nuclease domain of FokI (SEQ ID NO: 6).

5. The method according to claim 1, wherein the one or more PPR motifs are any group of motifs selected from 9 PPR motifs belonging to the p63 protein consisting of the amino acid sequence of SEQ ID NO: 1, 11 PPR motifs belonging to the GUN1 protein consisting of the amino acid sequence of SEQ ID NO: 2, 15 PPR motifs belonging to the pTac2 protein consisting of the amino acid sequence of SEQ ID NO: 3, 10 PPR motifs belonging to the DG1 protein consisting of the amino acid sequence of SEQ ID NO: 4, and 11 PPR motifs belonging to the GRP23 protein consisting of the amino acid sequence of SEQ ID NO: 5.