CN112175927A - Base editing tool and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a base editing tool and application thereof. The invention provides a fusion protein, which comprises an ecTadA-ecTadA dimer fragment and an xCas9n fragment, wherein the ecTadA-ecTadA dimer fragment comprises an ecTad fragment and an ecTadA fragment. The present invention provides a base editing tool that can efficiently recognize a site having a PAM sequence of NG, GAA, and GAT and can perform accurate and broader genome recognition by fusing a nuclear localization element and a DNA double strand break site binding protein to an xCas 9-based base editor. In addition, the fusion protein has high-efficiency and accurate single-base editing capacity, the purity of an editing product is high, and the fusion protein has a good industrialization prospect.

Description

Base editing tool and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a base editing tool and application thereof.

Background

The advent and development of CRISPR/Cas technology led to a revolutionary surge in the field of gene editing. Currently, the CRISPR/Cas9 system has been successfully applied to gene editing studies of multiple species, including knockout and knock-in of DNA, molecular labeling, and gene transcription regulation, among others. Under sgrna (single-stranded rna) guidance, Cas9 protein reaches a designated region and exerts nuclease activity to perform DNA double strand cleavage, resulting in DNA Double Strand Breaks (DSBs). The production of DSBs activates the body's own DNA Repair mechanisms, including Non-Homologous End joining Repair (NHEJ) and Homologous recombination Repair (HDR). Where NHEJ is the main repair pathway, the repair results in indels (indels) resulting in frameshift mutations (frameshift mutations), leading to gene knock-outs (knockouts); HDR-mediated repair can be achieved by knock-in (knock-in) in the presence of homologous templates, which is difficult to achieve with this approach due to the low efficiency and time and effort of HDR.

In response to the above problems, David Liu et al, harvard university, developed a Base editing tool (Base editor) capable of achieving single Base substitution without causing DSB by fusing Cas9nickase (Cas9n) having a nickase activity with a nucleotide deaminase. Currently, there are Cytosine Base Editors (CBE) and Adenine Base Editors (ABE) which can respectively realize C-to-T and A-to-G base substitution. When the CRISPR/Cas system targets a genome, a pro-spacer adjacent motif (PAM) is recognized, so that the PAM sequence becomes one of barriers for limiting the application of the existing base editing system. It has been studied that Cas9(SpCas9) from Streptococcus pyogenes (Streptococcus pyogenes) was modified to recognize sequences PAM as NG, GAA and GAT, and the modified Cas9 was named xCas 9. Compared with base editing tools (BE3 and ABE) based on SpCas9, the base editor (including xBE3 and xABE) based on xCas9 can identify more target sites on the genome, but the editing efficiency of xBE3 and xABE is relatively low, so that it has important application significance to improve the editing efficiency of xBE3 and xABE by fusing the nuclear localization element and the DNA double strand break binding protein.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a base editing tool and its use for solving the problems of the prior art.

To achieve the above and other related objects, according to one aspect of the present invention, there is provided a fusion protein comprising an ecTadA-ecTadA dimer fragment and an xCas9n fragment, wherein the ecTadA-ecTadA dimer fragment comprises an ecTad fragment and an ecTadA fragment.

In some embodiments of the invention, the amino acid sequence of said ecTadA fragment comprises:

a) an amino acid sequence shown as SEQ ID NO. 32; or the like, or, alternatively,

b) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.32, and having the function of the amino acid sequence defined in a), preferably capable of forming a dimer with an ecTadA fragment, the dimer having adenine deaminase activity.

In some embodiments of the invention, the amino acid sequence of said ecTadA fragment comprises:

c) an amino acid sequence shown as SEQ ID NO. 33; or the like, or, alternatively,

d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.33 and having the function of the amino acid sequence defined in c), preferably capable of forming a dimer with an ecTadA fragment, the dimer having adenine deaminase activity.

In some embodiments of the invention, the amino acid sequence of said xCas9n fragment comprises:

e) an amino acid sequence shown as SEQ ID NO. 34; or the like, or, alternatively,

f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.34 and having the function of the amino acid sequence defined in e), preferably capable of recognizing NG, GAA or GAT as PAM.

In some embodiments of the invention, the fusion protein comprises, in order from N-terminus to C-terminus, an ecTadA-ecTadA dimer fragment and an xCas9N fragment.

In some embodiments of the invention, the ecTadA-ecTadA dimer fragment comprises, in order from N-terminus to C-terminus, an ecTad fragment and an ecTadA fragment.

In some embodiments of the invention, the fusion protein further comprises a nuclear localization signal fragment, preferably the nuclear localization signal fragment is located at the N-terminus and/or the C-terminus of the ecTadA-ecTadA dimer fragment and the xCas9N fragment, preferably the amino acid sequence of the nuclear localization signal fragment is as shown in SEQ ID No.35, or SEQ ID No. 36.

In some embodiments of the invention, the fusion protein further comprises a flexible linker peptide fragment, preferably, the amino acid sequence of the flexible linker peptide fragment is as shown in SEQ ID No.37 or SEQ ID No. 38.

In some embodiments of the invention, the amino acid sequence of the fusion protein is set forth in SEQ ID No. 39.

In another aspect, the invention provides an isolated polynucleotide encoding the fusion protein.

In another aspect, the invention provides a construct comprising the isolated polynucleotide.

In another aspect, the invention provides an expression system comprising said construct or a genome into which said exogenous polynucleotide has been integrated.

In some embodiments of the invention, the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells, preferably from mouse cells, hamster cells, non-human primate cells, human cells, more preferably from mouse brain neuroma cells, chinese hamster ovary cells, african green monkey kidney cells, human osteosarcoma cells, human embryonic kidney cells, or human cervical cancer cells, more preferably from N2a cells, CHO cells, COS-7 cells, U2OS cells, HEK293FT cells, or Hela cells.

In another aspect, the invention provides the use of said fusion protein, said isolated polynucleotide, said construct or said expression system in gene editing.

In some embodiments of the invention, the use is in particular in gene editing in eukaryotes.

In another aspect, the invention provides a base editing system, which includes the fusion protein, and the base editing system further includes sgRNA.

In another aspect, the present invention provides a gene editing method, including: and (b) performing gene editing through the fusion protein or the base editing system.

Drawings

FIG. 1 shows a schematic diagram of the BPNLS-xBE vector and the editing efficiency detection thereof at partial test sites. Wherein a is a schematic diagram of an optimized BPNLS-xBE carrier; b is a Sanger sequencing peak diagram edited by the optimized BPNLS-xABE and the optimized pre-xABE at the endogenous gene locus; c, analyzing the editing efficiency of the BPNLS-xABE and xABE on the endogenous gene locus by applying Sanger sequencing; d is the standardization of the efficiency of editing endogenous gene sites by BPNLS-xABE and xABE.

FIG. 2 is a schematic diagram showing the editing efficiency of BPNLS-xABE simulated pathogenic sites in example 1 of the present invention. Wherein a is a Sanger sequencing peak diagram of a part of representative sites of BPNLS-xABE for pathogenic mutation simulation; b, analyzing and quantifying the simulation pathogenic mutation efficiency of BPNLS-xABE and xABE by applying Sanger sequencing; and c, simulating the mutation efficiency of two pathogenic sites simultaneously for BPNLS-xABE.

FIG. 3 is a schematic diagram showing the Wilson's disease-causing mutant cell line of example 2 of the present invention. Wherein a is Wilson disease pathogenic gene mutation site Atp7b^T1033APosition and sequence information in the gene, and sgRNA sequences for modeling and repairing the pathogenic mutation; b is Wilson disease pathogenic gene mutation site Atp7b^T1220MPosition and sequence information in the gene, and sgRNA sequences for modeling and repairing the pathogenic mutation; c, successfully constructing the Wilson disease pathogenic mutation site Atp7b by applying BPNLS-xABE^T1033ASanger sequencing peak plot of cell lines; d is to use BPNLS-Gam-xBE3 to successfully construct a mutant site Atp7b containing Wilson disease causing mutation^T1220MSanger sequencing peak plot of cell lines.

FIG. 4 shows the experiment of example 3 of the present invention using BPNLS-Gam-xBE3 and BPNLS-xABE for Atp7b respectively^T1033AAnd Atp7b^T1220MSchematic representation of the repair. Wherein a is BPNLS-Gam-xBE3 applied to pathogenic mutation site Atp7b containing Wilson's disease^T1033ARepair of mutation sites in cell linesSanger sequencing peak plot of (a); b is Wilson disease pathogenic mutation site Atp7b^T1033AQuantification in mutant and repair cells; c, applying BPNLS-xABE to the pathogenic mutation site Atp7b containing Wilson's disease^T1220MSanger sequencing peak diagrams of repair of mutation sites in cell lines; d is Wilson disease pathogenic mutation site Atp7b^T1220MQuantification in mutant and repair cells.

Detailed Description

The present inventors have made extensive exploratory studies and have provided a fusion protein which is a novel adenine base editing tool, and which can recognize NG, GAA and GAT sites and perform precise base editing, widening the targeting range of base editing, and have completed the present invention.

In a first aspect, the invention provides a fusion protein comprising an ecTadA-ecTadA dimer fragment and an xCas9n fragment, said ecTadA-ecTadA dimer fragment comprising an ecTad fragment and an ecTadA fragment. The fusion protein can use NG, GAA and GAT as PAM sequences, is matched with sgRNA in a target area, realizes efficient base editing of 4-7A-to-G at the 5' end of the sgRNA in the target area, and has higher editing efficiency and higher editing accuracy at the target site.

In the fusion protein provided by the present invention, the amino acid sequence of the ecTadA fragment may comprise: a) an amino acid sequence shown as SEQ ID NO. 32; or b) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.32 and having the function of the amino acid sequence defined in a). Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID NO.32 is obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids, or one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids are added to the N-terminal and/or C-terminal, and the polypeptide fragment has the function of the polypeptide fragment shown in SEQ ID NO.32, for example, a polypeptide fragment having a dimer activity capable of forming a dimer with an ecTadA fragment and having an adenine deaminase activity, more specifically, a polypeptide fragment having an adenine (enadine, A) deamination functions to produce hypoxanthine (I). The amino acid sequence in b) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID NO. 32. The ecTadA fragment is derived from Escherichia coli (Escherichia coli).

In the fusion protein provided by the present invention, the amino acid sequence of the ecTadA fragment may comprise: c) an amino acid sequence shown as SEQ ID NO. 33; or d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.33 and having the function of the amino acid sequence defined in c). Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown in SEQ ID NO.33 may be obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids, or may be obtained by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids to the N-terminus and/or the C-terminus, and may have a function of the polypeptide fragment shown in SEQ ID NO.33, for example, a polypeptide fragment having a function of forming a dimer with an ecTadA fragment and having an adenine deaminase activity, more specifically, adenine (adenine, A) deamination functions to produce hypoxanthine (I). The amino acid sequence in d) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID NO. 33. The ecTadA fragments are derived from e.coli (Escherichia coli) and obtained by artificial directed evolution.

In the fusion protein provided by the present invention, the amino acid sequence of the xCas9n fragment may include: e) an amino acid sequence shown as SEQ ID NO. 34; or f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.34 and having the function of the amino acid sequence defined in e). Specifically, the amino acid sequence in f) specifically refers to: the amino acid sequence shown in SEQ ID NO.34 may be obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids, or one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids may be added to the N-terminus and/or the C-terminus, and the polypeptide fragment having the function of the polypeptide fragment shown in SEQ ID NO.34 as an amino acid, for example, may be a polypeptide fragment capable of recognizing NG, GAA, or GAT as PAM, specifically may be a polypeptide fragment capable of recognizing NG, GAA, or GAT as PAM, and can be matched with sgRNA and ecTadA-ecTadA dimer fragments of specific target sites to realize base editing of A-to-G at 4-7 sites of the 5' end of the sgRNA in a target region. The amino acid sequence in f) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 34. Targeted recognition of the CRISPR/Cas9 system usually requires a pro-spacer adjacent motif (PAM) next to the target site, as one of the Cas9 enzymes most frequently used for genome editing, Cas9(SpCas9) from Streptococcus pyogenes (Streptococcus pyogenes) is only able to recognize PAM for the NGG sequence, which limits the range that can be targeted in the genome, whereas the xCas9n fragment of the present invention is derived from Streptococcus pyogenes (Streptococcus pyogenes) is able to recognize GAA, GAA or GAT sequence as PAM.

In the fusion protein provided by the invention, the substitution, deletion or addition can be conservative amino acid substitution. The "conservative amino acid substitution" may specifically refer to the case where an amino acid residue is substituted with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains should be known to those skilled in the art and may be, for example, families including, but not limited to, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). More specifically, conservative amino acid substitutions may include, but are not limited to, the particulars listed in the following table, where the numbers in table 1 (amino acid similarity matrix) indicate the degree of similarity between two amino acids, and where the numbers are greater than or equal to 0, they are considered conservative amino acid substitutions, and table 2 is an exemplary scheme of conservative amino acid substitutions.

TABLE 1

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	I	L	F	Y	W
																					W	-8	-7	-6	-2	-6	-5	-7	-7	-4	-5	-3	-3	2	-6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	-2	-2	-1	-1	7	10
																					F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	-1	0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
																					I	-2	-3	-2	-1	-1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5
M	-5	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
																					V	-2	-1	-1	-1	0	0	-2	-2	-2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
																					K	-5	-2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
																					Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
																					E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
																					T	-2	0	0	1	1	3
A	-2	1	1	1	2
																					S	0	1	1	1
P	-3	-1	6
																					G	-3	5
C	12

TABLE 2

The fusion protein provided by the invention can also comprise a nuclear localization signal fragment (NLS), and the nuclear localization signal fragment can generally interact with a nuclear vector, so that the protein can be transported into a nucleus. The nuclear localization signal fragment may be located at the N-terminus of the ecTadA-ecTadA dimer fragment, at the C-terminus of the xCas9N fragment, or between the ecTadA-ecTadA dimer fragment and the xCas9N fragment. The skilled person can generally select the position and specific sequence of suitable nuclear localization signal fragments, for example, the N-terminus of the ecTadA-ecTadA dimer fragment can be provided with a first nuclear localization signal fragment, the amino acid sequence of which can include the amino acid sequence shown in SEQ ID No. 35; as another example, the C-terminus of the xCas9n fragment may be provided with a second nuclear localization signal fragment, and the amino acid sequence of the second nuclear localization signal fragment may include the amino acid sequence shown as SEQ ID NO. 36.

The fusion protein provided by the invention can further comprise a flexible connecting peptide segment, wherein the flexible connecting peptide segment can be positioned at the N end of the ecTadA-ecTadA dimer segment, between the ecTadA segment and the ecTadA segment, between the ecTadA-ecTadA dimer segment and the xCas9N segment, or at the C end of the xCas9N segment. Those skilled in the art will generally select suitable flexible linker fragments for linking the polypeptide fragments, for example, a first flexible linker fragment may be provided between the ecTad fragment and the ecTadA fragment, and the amino acid sequence of said first flexible linker fragment may comprise the amino acid sequence shown in SEQ ID No. 37; for another example, a second flexible linker peptide segment may be disposed between the ecTadA-ecTadA dimer fragment and the xCas9n fragment, and an amino acid sequence of the second flexible linker peptide segment may include an amino acid sequence shown in SEQ ID No. 37; for another example, the C-terminus of the xCas9n fragment may be provided with a third flexible linker, and the amino acid sequence of the third flexible linker may include the amino acid sequence shown in SEQ ID No. 38.

In the fusion protein provided by the invention, the fusion protein may sequentially include an ecTadA-ecTadA dimer fragment and an xCas9N fragment from the N-terminal to the C-terminal, and the ecTadA-ecTadA dimer fragment may sequentially include an ecTadA fragment and an ecTadA fragment from the N-terminal to the C-terminal, and preferably may sequentially include an ecTadA fragment, a first flexible linker fragment and an ecTadA fragment. In a specific embodiment of the present invention, the fusion protein may sequentially include, from the N-terminal to the C-terminal, a first nuclear localization signal fragment, an ecTadA-ecTadA dimer fragment, a second flexible linker fragment, an xCas9N fragment, a third flexible linker fragment, and a second nuclear localization signal fragment, and an amino acid sequence of the fusion protein is shown in SEQ ID No. 39.

In a second aspect, the present invention provides an isolated polynucleotide encoding a fusion protein provided by the first aspect of the present invention.

In a third aspect, the invention provides a construct comprising an isolated polynucleotide provided in the second aspect of the invention. The construct can be generally constructed by inserting the isolated polynucleotide into a suitable expression vector, which can be selected by those skilled in the art, for example, including but not limited to, a pCMV expression vector, a pSV2 expression vector, a pGL3 expression vector, a pST1347 vector, a px330 vector, a pAAV vector, and the like.

In a fourth aspect, the invention provides an expression system comprising a construct or genome provided by the third aspect of the invention and integrated therein an exogenous isolated polynucleotide provided by the second aspect of the invention. The expression system can be a host cell that can express the fusion protein as described above, which can cooperate with the sgRNA such that the fusion protein can be targeted to the target region, enabling base editing of the target region. In another embodiment of the present invention, the host cell may be a eukaryotic cell and/or a prokaryotic cell, more specifically a mouse cell, hamster cell, non-human primate cell, human cell, etc., more specifically a mouse brain neuroma cell, chinese hamster ovary cell, african green monkey kidney cell, human osteosarcoma cell, human embryonic kidney cell, human cervical cancer cell, etc., more specifically N2a cell, CHO cell, COS-7 cell, U2OS cell, HEK293FT cell, Hela cell, etc.

In a fifth aspect, the invention provides the fusion protein provided by the first aspect of the invention, or the isolated polynucleotide provided by the second aspect of the invention, or the construct provided by the third aspect of the invention, or the expression system provided by the fourth aspect of the invention, for use in gene editing, preferably for use in gene editing of a eukaryote, particularly an metazoan, particularly including but not limited to humans, non-human primates, hamsters, mice, and the like. The uses may specifically include, but are not limited to, base editing from a to G (more specifically base editing from a-to-G at positions 4-7 of the 5' end of the sgRNA in the target region), editing of splice acceptor/donor sites to modulate RNA splicing, construction of models (e.g., disease models, cellular models, animal models, etc.) or treatment of human diseases using the present tools, and the like. In a specific embodiment of the invention, the use may specifically be a targetThe specific editing site of the gene from A to G can be ATP7b^M1169V(GenBank:NC_000013.11)、ATP7b^I1230V、ATP7b^W1353R、ATP7b^Y713C、STK11^L67P(GenBank:NC_000019.10)、ARHGEF18^T270A(GenBank:NC_000019.10)、BCS1L^V327A(GenBank:NC_000002.12)、BCS1L^M48V、AIFM1^D237G(GenBank:NC_000023.11)、ALPL^C201R(GenBank: NC-000001.11), and the like. In a specific embodiment of the invention, the application is specifically the construction or repair of a cell line, more specifically a cell line containing Wilson disease-causing mutation, or a PJ's syndrome-causing mutation, and the like, and the specific editing site can be Atp7b^T1033A、Atp7b^T1220M、STK11^D194N、STK11^R297KAnd the like. In another embodiment of the present invention, the object being edited may be an embryo, a cell, or the like.

In a sixth aspect, the invention provides a base editing system, including the fusion protein provided in the first aspect, the base editing system further including sgRNA. One skilled in the art can select an appropriate sgRNA targeting a specific site according to the targeted editing region of the gene. For example, the sequence of the sgRNA can be at least partially complementary to the target region, so that it can cooperate with the fusion protein to localize the fusion protein to the target region and allow base editing, specifically adenine deamination, i.e., the editing of adenine (a) to hypoxanthine (I), of a-to-G at positions 4-7 of the 5' end of the sgRNA in the target region. The base editing system provided by the invention greatly widens the targeted range of the genome, can take NG, GAA or GAT sequences as PAM, realizes the bases of A-to-G at 4-7 sites of the 5' end in the sgRNA target region, and has high mutation precision and low adjacent miss distance. In a specific embodiment of the invention, the application is specifically the construction or repair of a cell strain, more specifically a mutant cell strain containing Wilson disease, or a mutant cell strain containing PJ's syndrome. In a specific embodiment of the invention, the application is specifically the construction of cell lines, more specifically containing Wilson disease causing mutationThe specific editing site may be Atp7b, such as construction or repair of cell lines, or construction or repair of mutant PJ's syndrome cell lines^T1033A(GenBank:NC_000013.11)、Atp7b^T1220M(Genbank:NC_000013.11)、STK11^D194N(GenBank:NC_000019.10)、STK^R297K(GenBank: NC-000019.10), and the like. In another embodiment of the present invention, the object being edited may be an embryo, a cell, or the like.

The seventh aspect of the present invention provides a base editing method comprising: the gene editing is performed by the fusion protein provided by the first aspect of the present invention or the base editing system provided by the sixth aspect of the present invention. For example, the gene editing method may include: culturing the expression system provided by the fourth aspect of the present invention under appropriate conditions to express the fusion protein, which can base-edit the target region in the presence of the sgRNA targeting the target region to which it is mated. Methods for providing conditions under which the sgRNA exists should be known to those skilled in the art, and for example, an expression system capable of expressing the sgRNA, which may be a host cell including an expression vector containing a polynucleotide encoding the sgRNA or a host cell having the polynucleotide encoding the sgRNA integrated in a chromosome, may be cultured under appropriate conditions. In a specific embodiment of the present invention, the sgRNA and the fusion protein can be expressed in the same host cell, which is the target cell. In another embodiment of the invention, the gene editing is in vitro gene editing.

The present invention provides a base editing tool that can efficiently recognize a site having a PAM sequence of NG, GAA, and GAT and can perform accurate and broader genome recognition by fusing a nuclear localization element and a DNA double strand break site binding protein to an xCas 9-based base editor. In addition, the fusion protein has high-efficiency and accurate single-base editing capacity, the purity of an editing product is high, and the fusion protein has a good industrialization prospect.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The construction methods of the BPNLS-Gam-xBE3 vector and the BPNLS-xBE vector used in the examples are as follows: xBE3 and xABE were optimized by introducing the BPNLS and Gam elements in the xBE3 vector and the BPNLS elements in the xABE vector, respectively, xBE3 and xABE plasmids were from addge (#108380, # 108382). The sequences of the constructed BPNLS-Gam-xBE3 and BPNLS-xABE vectors are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2.

Example 1

In this example, the editing efficiency of BPNLS-xABE was verified in HEK293T cells using selected sgRNA sites of the endogenous gene, and the results are shown in fig. 1 and 2.

1.1 sgRNA plasmid construction

Selection of 10 endogenous Gene loci (including ATP7 b)^M1169V,ATP7b^I1230V,ATP7b^W1353R,ATP7b^Y713C,STK11^L67P,ARHGEF18^T270A,BCS1L^V327A,BCS1L^M48V,AIFM1^D237G,ALPL^C201RAnd the like), designing sgRNA according to a target sequence and synthesizing oligos, wherein the sequence of the sgRNA is shown as SEQ ID.3-22. The 5 'end of the upstream sequence of each sgNRA was added with an accg sequence and the 5' end of the downstream sequence was added with an aaac sequence, so that the upstream sequence of each sgRNA used for synthesis was: 5 '-accgXXXXXXXXXXXXXXXXXX (20nt) -3', downstream sequence: 5 '-aaacXXXXXXXXXXXXXXXXXX (20nt) -3'. After synthesis, the upstream and downstream sequences were annealed by a preset program (95 ℃, 5 min; 95 ℃ -85 ℃ at-2 ℃/s; 85 ℃ -25 ℃ at-0.1 ℃/s; hold at 4 ℃), the annealed product was ligated to pGL3-U6-sgRNA (Addgene #51133) vector linearized with BsaI (NEB: R0539L), the sequence of pGL3-U6-sgRNA vector being shown as SEQ ID.31. The linearization system of pGL3-U6-sgRNA vector is as follows: pGL3-U6-sgRNA 3. mu.g; buffer (NEB: R0539L) 6. mu.L; BsaI 2. mu.L; ddH2O supplementThe enzyme was digested overnight at 37 ℃ in a volume of 60. mu.L. The sgRNA annealing product was ligated to the linearized vector as follows: 1 μ L of T4 ligase buffer (NEB: M0202L), 20ng of linearized vector, 5 μ L of annealed oligo fragment (10 μ M), 0.5 μ L of T4 ligase (NEB: M0202L), and ddH2O filled to 10 μ L and ligated overnight at 16 ℃. And transforming the connected vector into escherichia coli, selecting bacteria for identification, carrying out sequencing confirmation, carrying out shake bacteria on positive clones, extracting plasmids (Axygene: AP-MN-P-250G), measuring the concentration, and storing for later use.

1.2 cell culture and transfection

HEK293T cells (purchased from ATCC) were inoculated in DMEM high-sugar medium (HyClone, SH30022.01B) supplemented with 10% FBS (v/v) containing 1% Penicillin Streptomycin (v/v) (Gibco) and cultured in a 37-degree cell incubator containing 5% CO 2. The cells used for transfection were inoculated in 12-well cell culture plates for culture on the first day, the cells were observed on the next day, and when the cells grew to a cell density of about 75%, the cells were optimally restored by changing the medium with fresh antibiotic-free DMEM medium containing 10% FBS for 2 hours. When in transfection, the dosage of plasmids transfected by each hole of the 12-hole plate is 1 mu g of BPNLS-Gam-xBE3 or BPNLS-xABE plasmid and 0.6 mu g of sgRNA plasmid respectively. The plasmids were mixed and diluted with 50. mu.l of Opti-MEM (Gibco,11058021) medium to prepare a reagent A, and the mixture was allowed to stand for 5 minutes. Meanwhile, 2. mu.l of Lipofectamine 2000 transfection reagent (Thermo,11668019) was diluted with 50. mu.l of Opti-MEM medium and mixed well as reagent B, and left to stand for 5 minutes. And mixing the reagent A and the reagent B, uniformly blowing, and standing for 20 minutes. After the standing is finished, the mixed reagent is dropwise added into 12-hole plate cells to be transfected and placed back to a 37-degree incubator for culture. The medium was changed to DMEM medium containing 10% FBS 6 hours after transfection. Fluorescence was observed under a microscope 48 hours after transfection, and cells harvested 72 hours after transfection were sorted for GFP positive cells using a flow cytometer. 5000-10000 GFP positive cells are collected for each sample as required, and the samples are cracked and genotyped by cell lysate after centrifugation. The main components of the cell lysate were 50mM KCl, 1.5mM MgCl2, 10mM Tris pH 8.0, 0.5% Nonidet P-40, 0.5% Tween 20, 100g/ml protease K.

1.3 detection of editing efficiency of optimized base editing tool at endogenous Gene locus

Designing a primer according to the experimental requirement, carrying out PCR amplification on a sequence near a target point by using the GFP positive cell lysis product of 1.2 as a template, carrying out gel electrophoresis on the amplification product to detect a target band, and then purifying. The purified PCR product is used for high-throughput deep sequencing or Sanger sequencing for the identification of editing efficiency. The system for amplification of the target site sequence is as follows: 2Xbuffer (Vazyme, P505) 25. mu.L; dNTP 1 u L; f (10 pmol/. mu.L) 1. mu.L; r (10 pmol/. mu.L) 1. mu.L; 1 mu L of template; 0.5. mu.L of DNA polymerase (Vazyme, P505); ddH2O was made up to 50. mu.L. The amplified product is purified after the band of interest is determined by electrophoresis gel according to the following steps: adding PCR-A reagent (Axygen: AP-PCR-250G) with three times of volume into the PCR product, uniformly mixing, adding into A purification column, centrifuging at 12000 r/min for 1 min, and discarding the waste liquid; adding 700 mu L of Buffer W2, centrifuging for 1 minute, and discarding the waste liquid; adding 700 mu L of Buffer W2, centrifuging for 1 minute, and discarding the waste liquid; the purified column was housed in a recovery header and idled at 12000 rpm for 1 minute; the collection tube was replaced with a new one (1.5 mL), and after standing for 2 minutes, 20. mu.L of water was added to the column for elution. The correlation results are shown in fig. 1 and 2.

Example 2

In this example, the optimized base editing system of the present invention was used to simulate Wilson's disease-causing gene-related mutation Atp7b in a human cell line^T1033AAnd Atp7b^T1220MAnd constructing mutant cell strains respectively containing the two mutations, wherein the method is realized by using BPNLS-Gam-xBE3, BPNLS-xABE and corresponding sgRNA, and the result is shown in figure 3.

2.1 sgRNA plasmid construction

Near the mutation site, the upstream sequences of mutant sgRNA Atp7b-27 and sgRNA Atp7b-16, Atp7b-27 were designed as follows: 5'-accgATTACCCATGGCGTCCCCAG-3' (SEQ id No. 23), the downstream sequence is: 5'-aaacCTGGGGACGCCATGGGTAAT-3' (SEQ ID.24). The sequence upstream of ATP7b-16 is: 5'-accgGATCACGGGGGACAACCGGA-3' (SEQ id.25), the downstream sequence is: 5'-aaacTCCGGTTGTCCCCCGTGATC-3' (SEQ ID. 26). After synthesizing oligos, sgRNA plasmid construction is carried out according to the method 1.1, and after sequencing and identifying the correct sequence, plasmids are extracted and stored for later use.

2.2 culture and transfection of cells

HEK293T cells were cultured and transfected as described in 1.2. Construct Atp7b^T1033AThe plasmids used by the mutant cell line are BPNLS-xABE and sgRNA ATP7b-27, and the dosage of the plasmids is 1 mu g and 0.5 mu g respectively. Construct Atp7b^T1220MThe plasmids used by the mutant cell line are BPNLS-Gam-xBE3 and sgRNA Atp7b-16, and the dosage of the plasmids is 1 mu g and 0.5 mu g respectively.

2.3 construction of a cell line containing Wilson's disease-causing mutant cells

After 72 hours of cell transfection, the cells were resuspended by trypsinization and sorted by flow cytometry. Selecting single cells with strong GFP positive, sorting to a 96-well plate, culturing for about two weeks in a 37-degree incubator, and selecting monoclonal cells under a microscope. And (3) selecting the monoclonal cells with good state for digestion and resuspension, numbering all the selected monoclonal cells, wherein one part is used for genotype identification, and the other part is subjected to passage amplification. After centrifugation of the cells for genotyping, the supernatant was discarded and the pellet at the bottom of the centrifuge tube was lysed with cell lysis buffer as described in 1.2. Primers were designed based on the target sequence and PCR amplification was performed using the cell lysate as template, as described in 1.3. And identifying target bands of the PCR amplification products by gel electrophoresis and then performing Sanger sequencing. Selection of pathogenic site Atp7b based on Sanger sequencing results peak plot^T1033AAnd Atp7b^T1220MAnd carrying out passage amplification and subsequent experiments on the completely mutated homozygous genotype monoclonal cells. The correlation results are shown in FIG. 3.

Example 3

In this example, the obtained homozygous mutant cell lines containing Wilson disease-causing mutation were treated with BPNLS-Gam-xBE3 and BPNLS-xABEAE of the present invention for Atp7b^T1033AAnd Atp7b^T1220MAnd (5) repairing. In this example, the repair of the mutation site was carried out using BPNLS-Gam-xBE3 and BPNLS-xABE plasmids in combination with the corresponding repair sgRNA, the structure of which is shown in FIG. 4.

3.1 sgRNA plasmid construction

In the vicinity of the mutation site, sgRNA was designed and repaired, and oligos were synthesized. Atp7b^T1033AAnd Atp7b^T1220MPathogenic factorThe repair sgrnas corresponding to the mutation sites are respectively: atp7 b-27-mut-corection sgRNA and Atp7 b-16-mut-coretionsgRNA. Atp7 b-27-mut-chromatography upstream sequence: 5'-accgATGGGTAATGGTGCCAGTCT-3' (SEQ ID.27), the downstream sequence being: 5'-aaacAGACTGGCACCATTACCCAT-3' (SEQ ID.28). Atp7 b-16-mut-chromatography upstream sequence: 5'-accgGTCCCCCGTGATCAGAACCA-3' (SEQ id No. 29), the downstream sequence is: 5'-aaac TGGTTCTGATCACGGGGGAC-3' (SEQ ID. 30). After synthesizing oligos, sgRNA plasmid construction is carried out according to the method 1.1, and after sequencing and identifying the correct sequence, plasmids are extracted and stored for later use.

3.2 cell culture and transfection

The gene which is constructed by the method 2.3 and contains the Wilson disease pathogenic mutation site Atp7b^T1033AAnd Atp7b^T1220MThe cell line (2) was cultured in a DMEM high-glucose medium containing 10% FBS and amplified. When repairing the pathogenic mutation, cell transfection was performed according to the method described in 1.2. Repair Atp7b^T1033AThe pathogenic site needs to transfect plasmids BPNLS-Gam-xBE3 and Atp7 b-27-mut-Cortion, the dosage of the plasmids is 1 mug and 0.6 mug respectively, the repair Atp7b^T1220MThe pathogenic site needs to be transfected with 1. mu.g of BPNLS-xABE and 0.6. mu.g of Atp7 b-16-mut-Cortion.

3.3 detection of repair efficiency

Cells were collected 72 hours after transfection and 10000 GFP strongly positive cells were sorted by a cell flow meter 5000-. Designing a primer to amplify the target fragment by PCR to identify the genotype. The main components of the cell lysate were 50mM KCl, 1.5mM MgCl2, 10mM Tris pH 8.0, 0.5% Nonidet P-40, 0.5% Tween 20, 100g/ml protease K. And (3) performing Sanger sequencing on the PCR amplification product after confirming a target band through electrophoresis gel, detecting whether the mutation site is repaired according to a Sanger sequencing peak diagram, and detecting the repair efficiency of the mutation site through quantification of the Sanger sequencing peak diagram. The correlation results are shown in fig. 4.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> Shanghai science and technology university

<120> a base editing tool and use thereof

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9201

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cctgtcaagg tatccccacg tcactctgtt tatttacatc gcaaggctgt accaccacgc 60

tgacccccgc aatcgacaag gcctgcggga tttgatctct tcaggtgtga ctatccaaat 120

tatgactgag caggagtcag gatactgctg gagaaacttt gtgaattata gcccgagtaa 180

tgaagcccac tggcctaggt atccccatct gtgggtacga ctgtacgttc ttgaactgta 240

ctgcatcata ctgggcctgc ctccttgtct caacattctg agaaggaagc agccacagct 300

gacattcttt accatcgctc ttcagtcttg tcattaccag cgactgcccc cacacattct 360

ctgggccacc gggttgaaat ctggtggttc ttctggtggt tctagcggca gcgagactcc 420

cgggacctca gagtccgcca cacccgaaag ttctggtggt tcttctggtg gttctgataa 480

aaagtattct attggtttag ccatcggcac taattccgtg ggctgggccg tgatcaccga 540

cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 600

caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 660

gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 720

agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 780

gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 840

ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 900

ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 960

gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 1020

gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 1080

cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 1140

aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 1200

cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggataccaa 1260

actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 1320

cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 1380

cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 1440

gctgtacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 1500

gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 1560

tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 1620

ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 1680

gaccttcgac aacggcatca tcccccacca gatccacctg ggagagctgc acgccattct 1740

gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 1800

cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 1860

ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgaga aggtggtgga 1920

caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 1980

caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 2040

gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 2100

ccagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 2160

gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 2220

ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 2280

caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 2340

gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 2400

cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 2460

ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 2520

gaagtccgac ggcttcgcca acagaaactt catccagctg atccacgacg acagcctgac 2580

ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 2640

cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 2700

ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 2760

ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 2820

gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 2880

cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 2940

cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 3000

tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 3060

ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 3120

gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 3180

cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 3240

aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 3300

cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 3360

gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 3420

cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 3480

gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 3540

caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 3600

gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 3660

cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 3720

gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 3780

cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 3840

gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 3900

gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 3960

gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 4020

agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 4080

gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgtgc tgcagaaggg 4140

aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 4200

gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 4260

gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 4320

cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 4380

agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 4440

cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 4500

ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 4560

tcagctggga ggcgactctg gtggttctac taatctgtca gatattattg aaaaggagac 4620

cggtaagcaa ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt 4680

cattgggaac aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga 4740

cgagaatgtc atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat 4800

acaggatagc aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa 4860

gaggaaagtc taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc 4920

tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 4980

accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 5040

tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 5100

gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 5160

gaaagaacca gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg 5220

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 5280

ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg 5340

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 5400

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 5460

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5520

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5580

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5640

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5700

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5760

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5820

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5880

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5940

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 6000

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 6060

agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 6120

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 6180

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 6240

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 6300

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 6360

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 6420

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 6480

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6540

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6600

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 6660

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 6720

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 6780

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 6840

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 6900

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6960

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 7020

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 7080

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 7140

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 7200

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 7260

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 7320

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac 7380

ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat 7440

gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 7500

gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 7560

cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 7620

tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 7680

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 7740

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 7800

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 7860

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 7920

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 7980

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 8040

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 8100

caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 8160

ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc 8220

cgctagagat ccgcggccgc taatacgact cactataggg agagccgcca ccatgaagcg 8280

caccgccgat ggttccgagt tcgaaagccc caaaaaaaag cgcaaggtcg ctaaaccagc 8340

aaaacgtatc aagagtgccg cagcggctta tgtgccacaa aaccgcgatg cggtgattac 8400

cgatattaaa cgcatcgggg atttacagcg cgaagcatca cgtctggaaa cggaaatgaa 8460

tgatgccatc gcggaaatta cggagaaatt tgcggcccgg attgcaccga ttaaaaccga 8520

tattgaaacc ctttcaaaag gcgttcaggg atggtgtgaa gcgaaccgcg acgaactgac 8580

gaacggcggc aaagtgaaga cggcgaatct tgtcaccggt gatgtatcgt ggcgggtccg 8640

tccaccatca gtaagtattc gtggtatgga tgcagtgatg gaaacgctgg agcgtcttgg 8700

cctgcaacgc tttattcgca cgaagcagga aatcaacaag gaagcgattt tactggaacc 8760

gaaagcggtc gcaggcgttg ccggaattac agttaaatca ggcattgagg atttttctat 8820

tattccattt gaacaggaag ccggtattag cggcagcgag actcccggga cctcagagtc 8880

cgctacaccc gaaagtagct cagagactgg cccagtggct gtggacccca cattgagacg 8940

gcggatcgag ccccatgagt ttgaggtatt cttcgatccg agagagctcc gcaaggagac 9000

ctgcctgctt tacgaaatta attggggggg ccggcactcc atttggcgac atacatcaca 9060

gaacactaac aagcacgtcg aagtcaactt catcgagaag ttcacgacag aaagatattt 9120

ctgtccgaac acaaggtgca gcattacctg gtttctcagc tggagcccat gcggcgaatg 9180

tagtagggcc atcactgaat t 9201

<210> 2

<211> 8792

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tctggtggtt ctcccaagaa gaagaggaaa gtctaaccgg tcatcatcac catcaccatt 60

gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 120

gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 180

aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 240

tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 300

tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgataccg tcgacctcta 360

gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 420

caattccaca caacatacga gccggaagca taaagtgtaa agcctagggt gcctaatgag 480

tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 540

cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 600

gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 660

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 720

agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 780

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 840

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 900

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 960

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 1020

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 1080

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 1140

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 1200

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 1260

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 1320

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 1380

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 1440

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 1500

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 1560

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 1620

tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 1680

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 1740

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 1800

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 1860

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 1920

gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 1980

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 2040

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 2100

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 2160

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 2220

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 2280

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 2340

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 2400

tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 2460

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 2520

gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc ctagggtcga 2580

ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg 2640

tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga 2700

ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac 2760

gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 2820

ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 2880

cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 2940

tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 3000

cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3060

acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3120

ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 3180

tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 3240

tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 3300

ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 3360

ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg actcactata 3420

gggccaccat gaagcgcacc gccgatggtt ccgagttcga aagccccaaa aaaaagcgca 3480

aggtcccgag agccgccacc atgtccgaag tcgagttttc ccatgagtac tggatgagac 3540

acgcattgac tctcgcaaag agggcttggg atgaacgcga ggtgcccgtg ggggcagtac 3600

tcgtgcataa caatcgcgta atcggcgaag gttggaatag gccgatcgga cgccacgacc 3660

ccactgcaca tgcggaaatc atggcccttc gacagggagg gcttgtgatg cagaattatc 3720

gacttatcga tgcgacgctg tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta 3780

tgattcactc ccgcattgga cgagttgtat tcggtgcccg cgacgccaag acgggtgccg 3840

caggttcact gatggacgtg ctgcatcacc caggcatgaa ccaccgggta gaaatcacag 3900

aaggcatatt ggcggacgaa tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc 3960

aggagatcaa ggcccagaaa aaagcacaat cctctactga cagcggcggc agcagcggcg 4020

gcagcagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa agtagcggcg 4080

gcagcagcgg cggcagctcc gaagtcgagt tttcccatga gtactggatg agacacgcat 4140

tgactctcgc aaagagggct cgggatgaac gcgaggtgcc cgtgggggca gtactcgtgc 4200

ttaacaatcg cgtaatcggc gaaggttgga atagggcgat cggactccac gaccccactg 4260

cacatgcgga aatcatggcc cttcgacagg gagggcttgt gatgcagaat tatcgactta 4320

tcgatgcgac gctgtacgtc acgtttgaac cttgcgtaat gtgcgcggga gctatgattc 4380

actcccgcat tggacgagtt gtattcggtg tccgcaacgc caagacgggt gccgcaggtt 4440

cactgatgga cgtgctgcat tacccaggca tgaaccaccg ggtagaaatc acagaaggca 4500

tattggcgga cgaatgtgcg gcgctgttgt gctacttttt tcgcatgccg aggcaggtgt 4560

tcaatgccca gaaaaaagca caatcctcta ctgacagcgg cggcagcagc ggcggcagca 4620

gcggcagcga gactcccggg acctcagagt ccgccacacc cgaaagtagc ggcggcagca 4680

gcggcggcag cgataaaaag tattctattg gtttagccat cggcactaat tccgtgggct 4740

gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 4800

ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa 4860

cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc 4920

ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct 4980

tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca 5040

tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 5100

tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 5160

tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 5220

acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 5280

aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 5340

agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 5400

tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 5460

tggccgagga taccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 5520

tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 5580

acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 5640

gcgcctctat gatcaagctg tacgacgagc accaccagga cctgaccctg ctgaaagctc 5700

tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 5760

gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 5820

ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 5880

tgctgcggaa gcagcggacc ttcgacaacg gcatcatccc ccaccagatc cacctgggag 5940

agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 6000

aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 6060

gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 6120

tcgagaaggt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 6180

tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 6240

tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 6300

ccttcctgag cggcgaccag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 6360

aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 6420

tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 6480

tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 6540

atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 6600

cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 6660

gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga 6720

caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatc cagctgatcc 6780

acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg 6840

atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc 6900

tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga 6960

acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 7020

gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 7080

aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 7140

atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 7200

tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 7260

ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 7320

agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 7380

acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 7440

agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc 7500

ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc 7560

tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga 7620

tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga 7680

tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg 7740

tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct 7800

tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 7860

ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 7920

gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 7980

ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 8040

agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 8100

ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 8160

agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 8220

ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 8280

tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 8340

gcgtgctgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 8400

tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 8460

ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 8520

agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 8580

gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 8640

tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 8700

gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 8760

cacggatcga cctgtctcag ctgggaggcg ac 8792

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

accgggcact gcggctggag gtgg 24

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaacccacct ccagccgcag tgcc 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

accggcggtc tcaagcacta ccta 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaactaggta gtgcttgaga ccgc 24

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

accggagctg cacatactag cccc 24

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaacggggct agtatgtgca gctc 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accggcagac ggcagtcact aggg 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaacccctag tgactgccgt ctgc 24

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

accggtcgca ggacagcttt tcct 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaacaggaaa agctgtcctg cgac 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

accggggaag ctgggtgaat ggag 24

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacctccat tcacccagct tccc 24

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

accggcggag actctggtgc tgtg 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

aaaccacagc accagagtct ccgc 24

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

accgggtcag aaataggggg tcca 24

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aaactggacc ccctatttct gacc 24

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

accggatcca ggtgctgcag aagg 24

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aaacccttct gcagcacctg gatc 24

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

accggtcact ccaggattcc aata 24

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aaactattgg aatcctggag tgac 24

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

accgattacc catggcgtcc ccag 24

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aaacctgggg acgccatggg taat 24

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

accggatcac gggggacaac cgga 24

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

aaactccggt tgtcccccgt gatc 24

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

accgatgggt aatggtgcca gtct 24

<210> 28

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

aaacagactg gcaccattac ccat 24

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

accggtcccc cgtgatcaga acca 24

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aaactggttc tgatcacggg ggac 24

<210> 31

<211> 4927

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60

cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300

tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata 360

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 420

tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa 480

aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 540

tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 600

gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa 660

ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc 720

ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc 780

gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt 840

tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc 900

ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc 960

aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag 1020

gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc 1080

aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct 1140

ccccaggggg atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 1200

tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 1260

gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 1320

tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 1380

ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 1440

agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 1500

agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 1560

acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 1620

acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 1680

gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 1740

tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 1800

gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 1860

agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga 1920

ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa 1980

tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag 2040

catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 2100

actcatcaat gtatcttagt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 2160

tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 2220

aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc 2280

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 2340

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 2400

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 2460

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 2520

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2580

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 2640

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2700

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2760

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2820

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 2880

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2940

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 3000

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 3060

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 3120

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 3180

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 3240

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 3300

tctggcccca gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca 3360

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 3420

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 3480

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 3540

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 3600

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 3660

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 3720

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 3780

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 3840

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 3900

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 3960

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 4020

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 4080

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 4140

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca 4200

ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 4260

gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 4320

caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 4380

cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 4440

tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 4500

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 4560

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 4620

ttaacgttta caatttccca ttcgccattc aggctgcgca actgttggga agggcgatcg 4680

gtgcgggcct cttcgctatt acgccagccc aagctaccat gataagtaag taatattaag 4740

gtacgggagg tacttggagc ggccgcaata aaatatcttt attttcatta catctgtgtg 4800

ttggtttttt gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac 4860

aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct 4920

atcgata 4927

<210> 32

<211> 167

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu

1 5 10 15

Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala

20 25 30

Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro

35 40 45

Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

50 55 60

Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

65 70 75 80

Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His

85 90 95

Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly

100 105 110

Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His

115 120 125

Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu

130 135 140

Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys

145 150 155 160

Lys Ala Gln Ser Ser Thr Asp

165

<210> 33

<211> 167

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu

1 5 10 15

Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala

20 25 30

Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala

35 40 45

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

50 55 60

Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

65 70 75 80

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His

85 90 95

Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly

100 105 110

Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His

115 120 125

Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu

130 135 140

Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys

145 150 155 160

Lys Ala Gln Ser Ser Thr Asp

165

<210> 34

<211> 1367

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

20 25 30

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

35 40 45

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

85 90 95

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

115 120 125

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

130 135 140

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

165 170 175

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

180 185 190

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

195 200 205

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

210 215 220

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

225 230 235 240

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

245 250 255

Leu Ala Glu Asp Thr Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

260 265 270

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

275 280 285

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

290 295 300

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

305 310 315 320

Ile Lys Leu Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

325 330 335

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

340 345 350

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

355 360 365

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

370 375 380

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ile Ile Pro His Gln Ile His Leu Gly

405 410 415

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

420 425 430

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

450 455 460

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Lys Val

465 470 475 480

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

485 490 495

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

515 520 525

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Asp Gln Lys

530 535 540

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

545 550 555 560

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

565 570 575

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

610 615 620

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

625 630 635 640

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

645 650 655

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

660 665 670

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

675 680 685

Asn Arg Asn Phe Ile Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

690 695 700

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

705 710 715 720

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

725 730 735

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

820 825 830

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

835 840 845

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

850 855 860

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

865 870 875 880

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

900 905 910

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

915 920 925

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

930 935 940

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

945 950 955 960

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

965 970 975

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

980 985 990

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

995 1000 1005

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1010 1015 1020

Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn

1025 1030 1035 1040

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1045 1050 1055

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val

1060 1065 1070

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1075 1080 1085

Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1090 1095 1100

Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1105 1110 1115 1120

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1125 1130 1135

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1140 1145 1150

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1155 1160 1165

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys

1170 1175 1180

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1185 1190 1195 1200

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1205 1210 1215

Gly Val Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1250 1255 1260

Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile

1265 1270 1275 1280

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1285 1290 1295

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1300 1305 1310

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr

1315 1320 1325

Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala

1330 1335 1340

Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1345 1350 1355 1360

Leu Ser Gln Leu Gly Gly Asp

1365

<210> 35

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 36

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Pro Lys Lys Lys Arg Lys Val

1 5

<210> 37

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1 5 10 15

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

<210> 38

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Ser Gly Gly Ser

1

<210> 39

<211> 1799

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Pro Arg Ala Ala Thr Met Ser Glu Val Glu Phe Ser His

20 25 30

Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp

35 40 45

Glu Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val

50 55 60

Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala

65 70 75 80

His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn

85 90 95

Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val

100 105 110

Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe

115 120 125

Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val

130 135 140

Leu His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile

145 150 155 160

Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg

165 170 175

Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser

180 185 190

Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser

195 200 205

Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser

210 215 220

Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu

225 230 235 240

Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu

245 250 255

Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly

260 265 270

Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly

275 280 285

Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val

290 295 300

Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg

305 310 315 320

Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala

325 330 335

Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val

340 345 350

Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys

355 360 365

Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala

370 375 380

Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser

385 390 395 400

Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly

405 410 415

Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly

420 425 430

Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro

435 440 445

Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys

450 455 460

Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu

465 470 475 480

Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys

485 490 495

Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys

500 505 510

Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu

515 520 525

Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp

530 535 540

Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys

545 550 555 560

Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu

565 570 575

Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly

580 585 590

Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu

595 600 605

Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser

610 615 620

Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg

625 630 635 640

Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly

645 650 655

Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe

660 665 670

Lys Ser Asn Phe Asp Leu Ala Glu Asp Thr Lys Leu Gln Leu Ser Lys

675 680 685

Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp

690 695 700

Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile

705 710 715 720

Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro

725 730 735

Leu Ser Ala Ser Met Ile Lys Leu Tyr Asp Glu His His Gln Asp Leu

740 745 750

Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys

755 760 765

Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp

770 775 780

Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu

785 790 795 800

Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu

805 810 815

Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ile Ile Pro His

820 825 830

Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp

835 840 845

Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu

850 855 860

Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser

865 870 875 880

Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp

885 890 895

Asn Phe Glu Lys Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile

900 905 910

Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu

915 920 925

Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu

930 935 940

Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu

945 950 955 960

Ser Gly Asp Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn

965 970 975

Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile

980 985 990

Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn

995 1000 1005

Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys

1010 1015 1020

Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val

1025 1030 1035 1040

Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu

1045 1050 1055

Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys

1060 1065 1070

Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn

1075 1080 1085

Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys

1090 1095 1100

Ser Asp Gly Phe Ala Asn Arg Asn Phe Ile Gln Leu Ile His Asp Asp

1105 1110 1115 1120

Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln

1125 1130 1135

Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala

1140 1145 1150

Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val

1155 1160 1165

Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala

1170 1175 1180

Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg

1185 1190 1195 1200

Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu

1205 1210 1215

Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr

1220 1225 1230

Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu

1235 1240 1245

Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln

1250 1255 1260

Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser

1265 1270 1275 1280

Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val

1285 1290 1295

Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile

1300 1305 1310

Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu

1315 1320 1325

Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr

1330 1335 1340

Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn

1345 1350 1355 1360

Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile

1365 1370 1375

Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe

1380 1385 1390

Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr

1395 1400 1405

Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu

1410 1415 1420

Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys

1425 1430 1435 1440

Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr

1445 1450 1455

Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1460 1465 1470

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1475 1480 1485

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg

1490 1495 1500

Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val

1505 1510 1515 1520

Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser

1525 1530 1535

Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly

1540 1545 1550

Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys

1555 1560 1565

Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu

1570 1575 1580

Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp

1585 1590 1595 1600

Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile

1605 1610 1615

Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg

1620 1625 1630

Met Leu Ala Ser Ala Gly Val Leu Gln Lys Gly Asn Glu Leu Ala Leu

1635 1640 1645

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys

1650 1655 1660

Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu

1665 1670 1675 1680

Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe

1685 1690 1695

Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1700 1705 1710

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1715 1720 1725

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe

1730 1735 1740

Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys

1745 1750 1755 1760

Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr

1765 1770 1775

Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser

1780 1785 1790

Pro Lys Lys Lys Arg Lys Val

1795

Claims (14)

1. A fusion protein comprising an ecTadA-ecTadA dimer fragment and an xCas9n fragment, said ecTadA-ecTadA dimer fragment comprising an ecTad fragment and an ecTadA fragment.

2. The fusion protein of claim 1, wherein the amino acid sequence of the ecTadA fragment comprises:

a) an amino acid sequence shown as SEQ ID NO. 32; or the like, or, alternatively,

b) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.32, and having the function of the amino acid sequence defined in a), preferably capable of forming a dimer with an ecTadA fragment, the dimer having adenine deaminase activity.

3. The fusion protein of claim 1, wherein the amino acid sequence of the ecTadA fragment comprises:

c) an amino acid sequence shown as SEQ ID NO. 33; or the like, or, alternatively,

d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.33 and having the function of the amino acid sequence defined in c), preferably capable of forming a dimer with an ecTadA fragment, the dimer having adenine deaminase activity.

4. The fusion protein of claim 1, wherein the amino acid sequence of the xCas9n fragment comprises:

e) an amino acid sequence shown as SEQ ID NO. 34; or the like, or, alternatively,

f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.34 and having the function of the amino acid sequence defined in e), preferably capable of recognizing NG, GAA or GAT as PAM.

5. The fusion protein of claim 1, comprising an ecTadA-ecTadA dimer fragment and an xCas9N fragment in that order from N-terminus to C-terminus;

and/or the ecTadA-ecTadA dimer fragment comprises an ecTad fragment and an ecTadA fragment in that order from the N-terminus to the C-terminus.

6. The fusion protein of claim 1, further comprising a nuclear localization signal fragment, preferably wherein the nuclear localization signal fragment is located N-terminal and/or C-terminal to the ecTadA-ecTadA dimer fragment and the xCas9N fragment, preferably wherein the amino acid sequence of the nuclear localization signal fragment is as set forth in SEQ ID No.35, or SEQ ID No. 36;

and/or, the fusion protein further comprises a flexible connecting peptide fragment, preferably, the amino acid sequence of the flexible connecting peptide fragment is shown in SEQ ID NO.37 or SEQ ID NO. 38.

7. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID No. 39.

8. An isolated polynucleotide encoding the fusion protein of any one of claims 1 to 7.

9. A construct comprising the isolated polynucleotide of claim 8.

10. An expression system comprising the construct or genome of claim 9 having integrated therein an exogenous polynucleotide of claim 8.

11. The expression system of claim 10, wherein the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells, preferably from mouse cells, hamster cells, non-human primate cells, human cells, more preferably from mouse brain neuroma cells, chinese hamster ovary cells, african green monkey kidney cells, human osteosarcoma cells, human embryonic kidney cells, or human cervical cancer cells, more preferably from N2a cells, CHO cells, COS-7 cells, U2OS cells, HEK293FT cells, or Hela cells.

12. Use of a fusion protein according to any one of claims 1 to 7, an isolated polynucleotide according to claim 8, a construct according to claim 9 or an expression system according to any one of claims 10 to 11 for gene editing, preferably eukaryotic gene editing.

13. A base editing system comprising the fusion protein of any one of claims 1 to 7, the base editing system further comprising a sgRNA.

14. A method of gene editing comprising: gene editing is performed by the fusion protein according to any one of claims 1 to 7 or the base editing system according to claim 13.

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