CN115093482A - High-precision gonad purine base editor and application thereof - Google Patents

Abstract

The invention provides a high-precision ABEs base editor and application, wherein a fusion protein comprises a first region and a second region from an N end to a C end, the first region comprises ABEs, and the ABEs comprise adenine deaminase or an enzymatic active component thereof and nCas 9; the second region comprises e18 protein; the fusion protein fuses the e18 protein and an ABEs base editing system, the ABEs base editor utilizes the e18 fused with the ABEs base editor to accelerate the degradation of nCas9-TadA of the ABEs base editor, the half-life period of the ABEs base editor is reduced, and therefore the accuracy is improved.

Description

High-precision gonad purine base editor and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-precision gonadal purine base editor and application thereof.

Background

The single-base editor is a gene editing system formed by fusion expression of Cas9 protein and deaminase (mainly comprising adenine deaminase and cytosine deaminase) and other proteins. The system can realize the conversion from one base pair to another base pair accurately and irreversibly without introducing DNA double-strand break and exogenous repair templates. Single base editors currently mainly include Adenine Base Editors (ABEs), Cytosine Base Editors (CBEs), guanine base editors (GCBEs), and dcbes and TALEDs that enable precise editing of mitochondrial genomes.

An Adenine Base Editor (ABEs) is a fusion protein consisting of nCas9(D10A), artificially-modified Adenine deaminase and the like. The protein can specifically recognize and bind to a target sequence under the guidance of sgRNA, and adenine (A) is deaminated to form creatinine (I), and then the I is converted into G, so that A-G conversion is realized. Because the base editing system has the characteristics of high-efficiency base editing efficiency, simple operation and the like, various versions of ABEs (mainly comprising ABE7.10, ABEmax, ABE8e and the like) are widely applied at present, and the development of the fields of life sciences (such as gene therapy, human disease models, biological medicines, disease generating mechanisms and the like) is greatly promoted. However, with the progress of research, researchers found that ABEs, CBEs and the like have problems such as too large editing window, genome-wide off-target effect (including Cas 9-dependent off-target effect and Cas 9-independent off-target effect).

Therefore, in terms of application, it is important to develop a base editor with higher accuracy for the development of the life science field. Research shows that DNA base editing ribonucleoprotein complex (RNP) formed by sgRNA and ABEs fusion protein can be rapidly degraded by protease in cells, so that the purity of an editor product can be remarkably promoted, and off-target effect can be reduced. However, in the practical application process, the synthesis cost of RNP is high, the storage difficulty is high, and the application prospect of the RNP is greatly limited. The ubiquitin-proteasome system (UPS), as the major pathway for degradation of proteins, is the most important mechanism for controlling protein levels. Ubiquitination involves three main steps: activation, binding and ligation. The protein degradation process mainly activates ubiquitin by E1 ubiquitin activating enzyme, then E2 ubiquitin conjugating enzyme binds ubiquitin transferred by E1 ubiquitin activating enzyme, finally attaches ubiquitin selectively to lysine, serine, threonine or cysteine residue of the protein of interest by E3 ligase. The E3 ligase can bind directly to the substrate and determine the specificity of the ubiquitin system. Rad18 is a RING-type E3 ubiquitin ligase that plays a critical role in DNA damage repair, and the E18 protein is a Rad18 variant protein with SAP domain removed.

Disclosure of Invention

The invention aims to construct a high-precision ABEs base editor, which fuses e18 protein with an ABEs base editing system, wherein the ABEs base editor utilizes e18 fused with the ABEs base editor to accelerate degradation of nCas9-TadA of the ABEs base editor, reduces half-life period of the ABEs base editor, improves precision, and has the advantages of lower price, simpler operation, easier storage and the like compared with a DNA base editing RNP compound, and RNP does not need to be synthesized in vitro.

The purpose of the invention is realized by the following technical scheme:

a fusion protein comprising, from N-terminus to C-terminus, a first region and a second region, the first region comprising ABEs comprising adenine deaminase or an enzymatically active component thereof and nCas 9; the second region comprises e18 protein; the fusion protein optionally comprises one or more linker amino acid sequences located in the first region and between the first region and the second region of the fusion protein.

As a preferable technical scheme of the invention: the ABEs are one of ABE7.10, ABEmax and ABE8e base editors.

As a preferred technical scheme of the invention: the fusion protein also includes a nuclear localization signal fragment.

As a preferred technical scheme of the invention: the amino acid sequence of the fusion protein is shown in SEQ ID NO.40 when the ABEs is ABEmax; the amino acid sequence of the fusion protein is shown in SEQ ID NO.41 when the ABEs are ABE8 e.

It is also an object of the present invention to provide a polynucleotide encoding the above fusion protein. The polynucleotide sequence comprises a first region encoding the ABEs and a second region encoding the e18 protein, and optionally one or more linker amino acid sequences located in the first region and between the first and second regions of the fusion protein.

As a preferred technical scheme of the invention: when the ABEs are ABEmax, the polynucleotide sequence is constructed in the following way: the gene sequence of ABEmax before gene modification is shown in SEQ ID NO. 2; the upstream primer sequence F1 of the e18 gene fragment 1 capable of effectively amplifying the target gene is shown as SEQ ID NO.3, and the downstream primer sequence R1 is shown as SEQ ID NO. 4; an upstream primer sequence F2 of the e18 gene fragment 2 capable of effectively amplifying the target gene is shown as SEQ ID NO.5, and a downstream primer sequence R2 is shown as SEQ ID NO. 6; the upstream primer sequence F3 of the e18 fragment gene sequence with the enzyme cutting sites which can be effectively amplified is shown as SEQ ID NO.7, the downstream primer sequence R3 is shown as SEQ ID NO.8, and the e18 gene sequence with double enzyme cutting sites is shown as SEQ ID NO. 9; the sequence obtained by connecting the fragment after the restriction of the enzyme SEQ ID NO.9 with the fragment after the restriction of the enzyme SEQ ID NO.2 is the polynucleotide sequence shown in SEQ ID NO. 1.

As a preferred technical scheme of the invention: when the ABEs is ABE8e, the polynucleotide sequence shown in SEQ ID NO.10 is constructed in the following manner; the gene sequence of ABE8e before gene modification is shown in SEQ ID NO. 11; the e18 gene sequence with double restriction sites is shown as SEQ ID NO.9, and the sequence obtained by connecting the fragment SEQ ID NO.9 with the fragment after restriction of SEQ ID NO.11 is shown as the polynucleotide sequence shown as SEQ ID NO. 10.

It is also an object of the present invention to provide a construct comprising the polynucleotide. The construct may be constructed by inserting the polynucleotide into an appropriate expression vector. The expression vector may be, but is not limited to, a pCMV expression vector, a pSV2 expression vector, and the like.

It is also an object of the present invention to provide an expression system comprising said construct or having integrated into its genome the exogenous polynucleotide as described above. The expression system can be a host cell that can express the fusion protein as described above, which can be coordinated with the sgRNA so that the fusion protein can be targeted to the target region, enabling base editing of the target region.

It is also an object of the invention to provide a use, in particular a use of said fusion protein and said polynucleotide and said construct or said expression system in gene editing for converting the base a to G.

It is a further object of the invention to provide a base editing system comprising the fusion protein and sgrnas, the fusion protein and the sgrnas cooperating to localize the fusion protein to a target region.

It is still another object of the present invention to provide a method for gene editing comprising gene editing by converting the base A into G using the fusion protein or the base editing system.

The beneficial effects are as follows:

the adenine base editor with high accuracy fuses a foreign protein e18 on the existing adenine base editor, and the gene sequences of the plasmids are shown as SEQ ID NO.1 and SEQ ID NO.10, namely ABEmax-e18 and ABE8e-e 18. The editing windows all have obvious accuracy, namely 5-7 bits and 1-9 bits respectively. Editing positive clonal cells of PCSK9 significantly increased LDL uptake, indicating its potential for gene therapy. Provides more possible improvement direction for the development of the follow-up precise base editor, and simultaneously increases the tool number of the editor.

The invention can shorten the half-life of ABEs protein, accelerate ABEs expression plasmids degraded in cells, increase the accuracy of ABEs and reduce off-target effect of ABEs by constructing the ABEs protein expression plasmids.

Drawings

FIG. 1a is a schematic representation of the ABEmax-e18 plasmid vector of the present invention;

FIG. 1b is a schematic representation of the ABE8e-e18 plasmid vector of the present invention;

FIG. 2 is a sequencing statistical plot of the sgRNA base editing efficiency of ABEmax and ABEmax-e18 at multiple endogenous sites according to the present invention;

FIG. 3 is a sequencing diagram of a positive PCSK9 editing clone of the present invention;

FIG. 4 is a comparison of LDL uptake by wild-type cells of the invention and positive PCSK9 editing clone cells;

fig. 5 is a sequence statistical chart of sgRNA base editing efficiencies of ABE8e and ABE8e-e18 at multiple endogenous sites according to the present invention.

Detailed Description

The present invention provides in a first aspect a fusion protein comprising, from N-terminus to C-terminus, a first region and a second region, the first region comprising ABEs comprising adenine deaminase or an enzymatically active component thereof and nCas 9; the second region comprises e18 protein; the fusion protein optionally comprises one or more linker amino acid sequences located in the first region and between the first region and the second region of the fusion protein. The fusion protein is subjected to base editing at a target site under the guidance of the sgRNA, the ABEs fragment is an existing ABEs base editor, and the degradation of nCas9-TadA of the ABEs base editor is accelerated by using an e18 sequence which is fused and expressed with the ABEs base editor, so that the half-life of the ABEs base editor is reduced, and the accuracy is improved.

In the fusion protein provided by the invention, the ABEs are one of ABE7.10, ABEmax and ABE8e base editors.

In the fusion protein provided by the invention, the amino acid sequence of the fusion protein is shown as SEQ ID NO.40 or SEQ ID NO. 41; or an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.40 or to SEQ ID NO.41 and having the function of the amino acid sequence defined by SEQ ID NO.40 or SEQ ID NO. 41. Specifically, the amino acid sequence having more than 80% sequence similarity with SEQ ID NO.40 or SEQ ID NO.41 specifically refers to: the polypeptide fragment is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the amino acid sequence shown in SEQ ID NO.40 or SEQ ID NO.41, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment shown in SEQ ID NO.40 or SEQ ID NO. 41. The sequence similarity, which generally refers to the percentage of amino acid residues in the sequences that are identical in comparison, can be calculated using computational software known in the art for the similarity of two or more sequences of interest, e.g., software from NCBI.

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.

In the fusion protein provided by the invention, the fusion protein can also comprise a nuclear localization signal segment, and the nuclear localization signal segment can generally interact with a nuclear carrier, so that the protein can be transported into a nucleus.

In a second aspect, the present invention provides a polynucleotide encoding the fusion protein described above.

The polynucleotide sequence provided by the invention is shown in SEQ ID NO.1 or SEQ ID NO. 10.

In the polynucleotide provided by the invention, the polynucleotide sequence shown as SEQ ID NO.1 is constructed in the following way; the gene sequence of ABEmax before gene modification is shown in SEQ ID NO. 2; the upstream primer sequence F1 of the e18 gene fragment 1 capable of effectively amplifying the target gene is shown as SEQ ID NO.3, and the downstream primer sequence R1 is shown as SEQ ID NO. 4; the upstream primer sequence F2 of the e18 gene fragment 2 capable of effectively amplifying the target gene is shown as SEQ ID NO.5, and the downstream primer sequence R2 is shown as SEQ ID NO. 6; the upstream primer sequence F3 of the e18 fragment gene sequence with the enzyme cutting sites which can be effectively amplified is shown as SEQ ID NO.7, the downstream primer sequence R3 is shown as SEQ ID NO.8, and the e18 fragment with double enzyme cutting sites is shown as SEQ ID NO. 9; the sequence obtained by connecting the fragment after the restriction of the enzyme SEQ ID NO.9 with the fragment after the restriction of the enzyme SEQ ID NO.2 is the polynucleotide sequence shown in SEQ ID NO. 1.

In the polynucleotide provided by the invention, the polynucleotide sequence shown as SEQ ID NO.10 is constructed in the following way; the gene sequence of ABE8e before gene modification is shown in SEQ ID NO. 11; the sequence obtained by connecting the fragment SEQ ID NO.9 with the fragment obtained by enzyme digestion of the fragment SEQ ID NO.11 is shown as SEQ ID NO. 10.

In a third aspect, the invention provides a construct comprising the polynucleotide. The construct may be constructed by inserting the polynucleotide into an appropriate expression vector. One skilled in the art can select an appropriate expression vector, for example, the expression vector can be, but is not limited to, a pCMV expression vector, a pSV2 expression vector, and the like.

In a fourth aspect, the invention provides an expression system comprising the construct or the genome into which the exogenous polynucleotide has been integrated. The expression system can be a host cell that can express the fusion protein as described above, which can be coordinated with the sgRNA so 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, a human cell, etc., more specifically a mouse brain neuroma cell, a human embryonic kidney cell, a human cervical cancer cell, a human colon cancer cell, a human osteosarcoma cell, etc.

A fifth aspect of the invention provides a use of said fusion protein and said polynucleotide and said construct or said expression system in gene editing. Preferably eukaryotic organisms, in particular metazoan, in particular including but not limited to humans, mice, etc. The use specifically includes, but is not limited to, base editing from A to G, etc., which can be applied to edit a splice acceptor/donor site to regulate RNA splicing, and can also be used for constructing a model (e.g., a disease model, a cell model, an animal model, etc.) or treating human diseases, etc. In one embodiment of the present invention, the object being edited may be an embryo, a cell, or the like.

The sixth aspect of the invention provides a base editing system, which includes the fusion protein and sgRNA. One skilled in the art can select an appropriate sgRNA targeting a specific site according to the targeted editing region of a gene. For example, the sequence of the sgRNA can be generally at least partially complementary to the target region, such that it can cooperate with the fusion protein to localize the fusion protein to the target region, resulting in base editing in the target region, which is the conversion of base a to G.

The seventh aspect of the present invention provides a method for gene editing, comprising performing gene editing by converting the base a into G using the fusion protein or the base editing system. 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, wherein the fusion protein can perform base editing on the target region in the presence of sgRNA targeting the target region in cooperation with the fusion protein. Methods for providing conditions under which the sgrnas exist should be known to those skilled in the art, and for example, an expression system capable of expressing the sgrnas, which may be a host cell including an expression vector containing a polynucleotide encoding the sgrnas, or a host cell having a polynucleotide encoding the sgrnas chromosomally integrated therein, may be cultured under appropriate conditions. In a specific embodiment of the invention, the sgRNA and the fusion protein can be expressed in the same host cell, which can be a target cell. In another embodiment of the invention, the gene editing is in vitro gene editing.

The embodiments of the present invention are described below with specific examples, 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 its several details are capable of modifications and variations in various obvious respects, all without departing from the spirit of the 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 is 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 herein can be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples herein, in keeping with the knowledge of one skilled in the art and 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.

Example 1

Construction of ABEmax-e18 and ABE8e-e18 plasmids

Designing and synthesizing a primer sequence aiming at an e18 sequence, obtaining an e18 fragment with a restriction enzyme site by a PCR (polymerase chain reaction) in vitro amplification method, carrying out restriction enzyme on the fragment, and respectively connecting the fragment with ABEmax and ABE8e fragments which are similarly restricted to obtain ABEmax-e18 plasmids and ABE8e-e18 plasmids, wherein the main elements of the vector sequentially comprise: adenine deaminase, nCas9, e18, nuclear localization signal. The editor plasmid and the sgRNA plasmid act together to obtain characteristic information such as editing activity and the like. After the constructed plasmid is sequenced and verified, extracting a target plasmid, carrying out ethanol precipitation, purifying the expression vector with a certain concentration, and constructing a plasmid map as shown in figure 1.

The invention constructs the ABEs expression plasmid which can shorten the half-life period of ABEs protein, accelerate the degradation of the ABEs protein in cells, increase the accuracy of the ABEs and reduce the off-target effect of the ABEs.

The fusion protein sequentially comprises deaminase, nCas9 and an e18 fragment.

Example 2

Design of sgRNA sequence and plasmid construction

sgRNA sequences that can be used on human cells were designed and synthesized. Synthesizing the designed sgRNA sequence; the DNA sequences of the single-stranded sgRNAs form a plurality of oligonucleotide chains of the sgRNAs at different sites after annealing respectively; the oligonucleotide was then ligated into the sgRNA backbone plasmid vector. The sequences of these sgrnas are:

SgRNA-1 sequence: 5-GAATACTAAGCATAGACTCC-3

SgRNA-2 sequence: 5-GTAAACAAAGCATAGACTGA-3

SgRNA-3 sequence: 5-GAACACAAAGCATAGACTGC-3

SgRNA-4 sequence: 5-GATGAGATAATGATGAGTCA-3

SgRNA-5 sequence: 5-GACAAACCAGAAGCCGCTCC-3

SgRNA-6 sequence: 5-GGGAATAAATCATAGAATCC-3

SgRNA-7 sequence: 5-GGAACACAAAGCATAGACTG-3

SgRNA-8 sequence: 5-GCACCTACCTCGGGAGCTGA-3

SgRNA-9 sequence: 5-GGAATCCCTTCTGCAGCACC-3

SgRNA-10 sequence: 5-TCAGAAAGTGGTGGCTGGTG-3

SgRNA-11 sequence: 5-GGCCCAGACTGAGCACGTGA-3

SgRNA-12 sequence: 5-ATATTTGCATTGAGATAGTG-3

SgRNA-13 sequence: 5-GTCATCTTAGTCATTACCTG-3

SgRNA-14 sequence: 5-GAAGATAGAGAATAGACTGC-3

After sequencing verification of the constructed sgRNA expression vector, extracting a target plasmid, performing ethanol precipitation, and purifying the sgRNA expression vector with a certain concentration.

Example 3

Co-transfection of ABEs plasmid and sgRNA expression vector

3-1ABEmax and co-transfection of ABEmax-e18 plasmid with sgRNA expression vector

Plating HEK293T cells, introducing ABEmax and ABEmax-e18 and sgRNA expression vectors into the cells in a liposome transfection mode when the cells are about 80% long, after transfection is carried out for 72 hours, extracting genomes of all groups of cells, carrying out PCR reaction by using specific primers for detecting mutation efficiency, sending obtained PCR products to sequencing, evaluating editing efficiency of sgRNA sites through analysis of sequencing peak diagrams, and editing windows of editors; FIG. 2 shows the sgRNA1 sequences corresponding to SEQ ID NO.12 and SEQ ID NO.13 obtained above; sgRNA2 sequences corresponding to SEQ ID NO.14 and SEQ ID NO. 15; sgRNA3 sequences corresponding to SEQ ID NO.16 and SEQ ID NO. 17; sgRNA4 sequences corresponding to SEQ ID No.18 and SEQ ID No. 19; sgRNA5 sequences corresponding to SEQ ID NO.20 and SEQ ID NO. 21; sgRNA6 sequences corresponding to SEQ ID No.22 and SEQ ID No. 23; the sgRNA7 sequences corresponding to SEQ ID NO.24 and SEQ ID NO.25 can effectively guide cas9 protein to edit target sites in cells and obtain the characteristics of an editing window, the site range in the window editable by ABEmax is 3-8 sites, and the site range in the window editable by ABEmax-e18 is 5-7 sites.

Cotransfection of 3-2ABE8e and ABE8e-e18 plasmids with sgRNA expression vectors

Similarly, HEK293T cells are plated, ABE8e, ABE8e-e18 and sgRNA expression vectors are introduced into the cells in a liposome transfection mode when the cells are about 80% long, genomes of all groups of cells are extracted after transfection for 72 hours, then PCR reaction is carried out by using specific primers for detecting mutation efficiency, obtained PCR products are sent to sequencing, editing efficiency of sgRNA sites is evaluated through analysis of sequencing peak diagrams, and an editing window of an editor is obtained; FIG. 5 shows the sgRNA1 sequences corresponding to SEQ ID NO.12 and SEQ ID NO.13 obtained above; sgRNA2 sequences corresponding to SEQ ID No.14 and SEQ ID No. 15; sgRNA3 sequences corresponding to SEQ ID No.16 and SEQ ID No. 17; sgRNA4 sequences corresponding to SEQ ID No.18 and SEQ ID No. 19; sgRNA6 sequences corresponding to SEQ ID No.22 and SEQ ID No. 23; sgRNA9 sequences corresponding to SEQ ID No.28 and SEQ ID No. 29; sgRNA10 sequences corresponding to SEQ ID No.30 and SEQ ID No. 31; sgRNA11 sequences corresponding to SEQ ID NO.32 and SEQ ID NO. 33; sgRNA12 sequences corresponding to SEQ ID NO.34 and SEQ ID NO. 35; sgRNA13 sequences corresponding to SEQ ID No.36 and SEQ ID No. 37; the sgRNA14 sequences corresponding to SEQ ID NO.38 and SEQ ID NO.39 can effectively guide the cas9 protein to edit a target site in a cell and obtain the characteristics of an editing window, the site range in the editable window of ABE8e is 1-14, and the site range in the editable window of ABE8e-e18 is 1-9. E.g., the range of sites and the range of efficiencies within the editable window, see fig. 2.

Co-transfection of 3-3ABEmax-e18 plasmid with PCSK9-sgRNA expression vector

And (3) recovering HepG2 cells, washing the cells for 2-3 times by using PBS when the cells are nearly full, removing the supernatant, adding an electrotransfection buffer solution, adding the ABEmax-e18 plasmid and the sgRNA expression plasmid into the cells and the buffer solution in proportion, gently mixing the mixture by using a pipette, and gently sucking the mixture into the pipette with a special gun head. It is inserted into a pipette stand with an electrode cup into which the electric transfer buffer is added, the program is set up on the apparatus and then pressed to start. After the electric shock is finished and the mixture is kept still for 2 minutes, the mixture in the electric transfer gun is transferred into a cell culture dish. Finally, the cell culture dish is placed in a carbon dioxide incubator at 37 ℃ for culture. After 12 hours of incubation, the medium was changed. The results in fig. 3 show that the sgRNA8 sequences corresponding to SEQ ID No.26 and SEQ ID No.27 obtained above can effectively guide the cas9 protein to edit the target site in HepG2 cells, and see fig. 3.

Example 4 preparation of PCSK9 edited Positive clone HepG2 cells

After electrotransfection for 72h, the HepG2 cells were plated on 100mm cell culture dishes by limiting dilution method using pancreatin-digested cells, and the cell culture solution was changed once for 2-3 days. After the cell clones grow after 8-10 days, uniformly marking the cell clones under a fluorescence microscope, and then picking the marked clones into a 24-hole cell culture plate for subsequent culture. After 2-3 days, when the cells in the 24-well plate grow to a certain confluency, one half of the cells are cracked by NP40 lysate and then the PCSK9 fixed-point base editing event is further verified and determined by a PCR sequencing method, as shown in figure 4.

Experimental example 5, Positive PCSK9 site-directed base editing HepG2 cell in vitro LDL uptake assay HepG2 cells obtained above were cultured with DMEM, 5% FBS, 1% double antibody, 1mM sodium pyroguvate 1% glutamine, 1% non-essential amino acids. The obtained cells were seeded at a certain density in 6-well cell culture plates. When the cell density reached 70%, Dil-LDL was mixed at a ratio of 1: 100 dilution and cell incubation for 3 hours; the supernatant was then discarded, washed three times with PBS, cells were fixed in plates for 2h by adding 4% paraformaldehyde solution, and the samples were washed three times with PBS for 5 minutes each. Cells were permeabilized for 10 minutes with 0.5% triton X-100 (in PBS). PBS was washed three times for 5 minutes each. 0.5ug/ml DAPI (in PBS) was added for 10 min of staining. Wash three times with PBS. Observed under a fluorescent microscope. The site-specific edited cells were able to take up more LDL than the fluorescence intensity results of the unedited control cells, see fig. 5.

In conclusion, the invention realizes that the e18(rad18 gene removes SAP structure domain) gene sequence shown in SEQ ID NO.9 is fused to the commonly used ABEmax and ABE8e adenine base editor with the highest editing activity by using the action of the exogenous protein, so that the editor with more accurate editing window and lower editing activity is obtained, and a new direction is provided for further improving the editor subsequently.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described 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> Chongqing begonia Biotechnology research institute Co., Ltd

<120> high-precision gonadal purine base editor and application thereof

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ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

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tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc 1560

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 1620

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 1680

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1740

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1800

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1860

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1920

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1980

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 2040

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 2100

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 2160

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 2220

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 2280

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 2340

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 2400

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 2460

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 2520

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2580

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2640

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2700

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2760

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2820

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2880

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2940

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 3000

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 3060

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 3120

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 3180

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 3240

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 3300

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 3360

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 3420

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 3480

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3540

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3600

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3660

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3720

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3780

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3840

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3900

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3960

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 4020

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 4080

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 4140

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 4200

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 4260

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 4320

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 4380

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 4440

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 4500

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4560

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4620

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4680

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4740

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4800

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4860

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4920

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4980

cggcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 5040

tacggcggct tcgtgagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 5100

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 5160

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 5220

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 5280

aagagaatgc tggcctctgc cagattcctg cagaagggaa acgaactggc cctgccctcc 5340

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 5400

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 5460

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 5520

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5580

cacctgttta ccctgaccaa tctgggagcc cctcgggcct tcaagtactt tgacaccacc 5640

atcgaccgga aggtgtaccg gagcaccaaa gaggtgctgg acgccaccct gatccaccag 5700

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 5760

ggctcaaaaa gaaccgccga cggcagcgaa ttcagcacag ggagcatggg aatggactcc 5820

ctggccgagt ctcggtggcc tccgggcctg gcagtcatga agacaataga tgatttgctg 5880

cggtgtggaa tttgcttcga gtatttcaac attgcaatga taatacctca gtgttcacat 5940

aactactgct ctctctgtat aagaaaattt ctgtcctata aaactcagtg tccaacttgc 6000

tgtgtgactg tcacagagcc ggatctgaaa aataaccgca tattagatga actggtaaaa 6060

agcttgaatt ttgcacggaa tcatctgctg cagtttgctt tagagtcacc agccaaatct 6120

cctgcttctt cctcttcaaa gaatcttgct gtcaaagtat atactcctgt agcctccaga 6180

cagtctttaa agcaggggag caggttaatg gataatttct tgatcagaga aatgagtggt 6240

tctacatcag agttgttgat aaaagaaaat aaaagcaaat tcagccctca aaaagaggcg 6300

agccctgctg caaagaccaa agagacacgt tctgtagaag agatcgctcc agatccctca 6360

gaggctaagc gtcctgagcc accctcgaca tccactttga aacaagttac taaagtggat 6420

tgtcctgttt gcggggttaa cattccagaa agtcacatta ataagcattt agacagctgt 6480

ttatcacgcg aagagaagaa ggaaagcctc agaagttctg ttcacaaaag gaagccgcac 6540

atgtacaatg cccaatgcga tgctttgcat cctaaatcag ctgctgaaat agttcgagaa 6600

atcgaaaata tagagaagac taggatgcgt cttgaagcta gtaaactcaa tgaaagtgta 6660

atggttttta caaaggacca aacagaaaag gaaatagatg aaatccacag taaatatcgt 6720

aaaaaacata agagtgaatt tcagcttctg gtggatcagg ctagaaaagg atacaagaaa 6780

attgctggaa tgtcacaaaa aacagtaaca ataacaaaag aagatgaatc tacagaaaag 6840

ctatcttctg tatgcatggg acaggaagat aatatgacct cagtaacaaa ccacttttct 6900

caatcaaagc tggactcccc agaggaattg gaacctgaca gagaagagga ttcttctagc 6960

tgtattgata ttcaagaagt tctttcttca tcagaatcag attcatgcaa tagttccagt 7020

tcagacatca taagagatct tttagaagaa gaggaagcct gggaagcatc acataaaaac 7080

gatcttcaag acacagaaat aagtccaaga cagaatcgcc gcacaagagc cgctgaaagt 7140

gctgagattg aaccaagaaa caagcgtaat aggaatgaaa aaagaaccgc cgacggcagc 7200

gagttcgagc ccaagaagaa gaggaaagtc caaccggtca tcatcaccat caccattgag 7260

tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 7320

cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 7380

atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 7440

ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 7500

gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct 7560

agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 7620

ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga 7680

gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 7740

gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 7800

cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 7860

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 7920

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 7980

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 8040

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 8100

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 8160

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 8220

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 8280

actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 8340

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 8400

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 8460

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 8520

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 8580

tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa gggattttgg 8640

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 8700

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 8760

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 8820

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 8880

gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 8940

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 9000

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 9060

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 9120

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 9180

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 9240

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 9300

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 9360

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 9420

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 9480

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 9540

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 9600

tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 9660

acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 9720

aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc 9780

tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 9840

tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 9900

acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 9960

ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 10020

cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 10080

ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 10140

taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 10200

acttggcagt acatcaagtg tatc 10224

<210> 2

<211> 8811

<212> DNA

<213> Artificial sequence

<400> 2

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420

gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga agtcgagttt 480

agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga 540

gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac 600

aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga 660

ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca 720

tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca 780

cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg 840

aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc 900

gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc 960

gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc 1020

gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac 1080

gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg 1140

cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc 1200

atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg 1260

gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg 1320

atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac 1380

gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac 1440

cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc 1500

tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc 1560

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 1620

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 1680

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1740

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1800

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1860

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1920

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1980

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 2040

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 2100

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 2160

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 2220

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 2280

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 2340

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 2400

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 2460

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 2520

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2580

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2640

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2700

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2760

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2820

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2880

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2940

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 3000

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 3060

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 3120

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 3180

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 3240

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 3300

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 3360

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 3420

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 3480

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3540

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3600

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3660

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3720

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3780

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3840

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3900

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3960

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 4020

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 4080

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 4140

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 4200

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 4260

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 4320

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 4380

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 4440

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 4500

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4560

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4620

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4680

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4740

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4800

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4860

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4920

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4980

cggcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 5040

tacggcggct tcgtgagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 5100

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 5160

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 5220

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 5280

aagagaatgc tggcctctgc cagattcctg cagaagggaa acgaactggc cctgccctcc 5340

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 5400

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 5460

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 5520

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5580

cacctgttta ccctgaccaa tctgggagcc cctcgggcct tcaagtactt tgacaccacc 5640

atcgaccgga aggtgtaccg gagcaccaaa gaggtgctgg acgccaccct gatccaccag 5700

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 5760

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa 5820

ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt 5880

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5940

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 6000

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 6060

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 6120

ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 6180

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 6240

gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 6300

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 6360

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 6420

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 6480

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 6540

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 6600

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 6660

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6720

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 6780

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6840

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6900

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6960

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 7020

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 7080

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 7140

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga 7200

acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 7260

tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 7320

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7380

catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7440

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7500

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7560

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7620

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7680

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7740

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7800

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7860

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7920

cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7980

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 8040

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 8100

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 8160

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 8220

atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 8280

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 8340

gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa 8400

gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 8460

aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 8520

gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 8580

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8640

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8700

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8760

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c 8811

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<400> 3

atggactccc tggccgagtc t 21

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

cggcttcctt ttgtgaacag 20

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence

<400> 5

ctgttcacaa aaggaagccg cacatgtaca atgcccaatg cg 42

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<400> 6

attcctatta cgcttgtttc ttgg 24

<210> 7

<211> 45

<212> DNA

<213> Artificial sequence

<400> 7

gaattcagca cagggagcat gggaatggac tccctggccg agtct 45

<210> 8

<211> 86

<212> DNA

<213> Artificial sequence

<400> 8

accggttgga ctttcctctt cttcttgggc tcgaactcgc tgccgtcggc ggttcttttt 60

tcattcctat tacgcttgtt tcttgg 86

<210> 9

<211> 1451

<212> DNA

<213> Artificial sequence

<400> 9

gaattcagca cagggagcat gggaatggac tccctggccg agtctcggtg gcctccgggc 60

ctggcagtca tgaagacaat agatgatttg ctgcggtgtg gaatttgctt cgagtatttc 120

aacattgcaa tgataatacc tcagtgttca cataactact gctctctctg tataagaaaa 180

tttctgtcct ataaaactca gtgtccaact tgctgtgtga ctgtcacaga gccggatctg 240

aaaaataacc gcatattaga tgaactggta aaaagcttga attttgcacg gaatcatctg 300

ctgcagtttg ctttagagtc accagccaaa tctcctgctt cttcctcttc aaagaatctt 360

gctgtcaaag tatatactcc tgtagcctcc agacagtctt taaagcaggg gagcaggtta 420

atggataatt tcttgatcag agaaatgagt ggttctacat cagagttgtt gataaaagaa 480

aataaaagca aattcagccc tcaaaaagag gcgagccctg ctgcaaagac caaagagaca 540

cgttctgtag aagagatcgc tccagatccc tcagaggcta agcgtcctga gccaccctcg 600

acatccactt tgaaacaagt tactaaagtg gattgtcctg tttgcggggt taacattcca 660

gaaagtcaca ttaataagca tttagacagc tgtttatcac gcgaagagaa gaaggaaagc 720

ctcagaagtt ctgttcacaa aaggaagccg cacatgtaca atgcccaatg cgatgctttg 780

catcctaaat cagctgctga aatagttcga gaaatcgaaa atatagagaa gactaggatg 840

cgtcttgaag ctagtaaact caatgaaagt gtaatggttt ttacaaagga ccaaacagaa 900

aaggaaatag atgaaatcca cagtaaatat cgtaaaaaac ataagagtga atttcagctt 960

ctggtggatc aggctagaaa aggatacaag aaaattgctg gaatgtcaca aaaaacagta 1020

acaataacaa aagaagatga atctacagaa aagctatctt ctgtatgcat gggacaggaa 1080

gataatatga cctcagtaac aaaccacttt tctcaatcaa agctggactc cccagaggaa 1140

ttggaacctg acagagaaga ggattcttct agctgtattg atattcaaga agttctttct 1200

tcatcagaat cagattcatg caatagttcc agttcagaca tcataagaga tcttttagaa 1260

gaagaggaag cctgggaagc atcacataaa aacgatcttc aagacacaga aataagtcca 1320

agacagaatc gccgcacaag agccgctgaa agtgctgaga ttgaaccaag aaacaagcgt 1380

aataggaatg aaaaaagaac cgccgacggc agcgagttcg agcccaagaa gaagaggaaa 1440

gtccaaccgg t 1451

<210> 10

<211> 9630

<212> DNA

<213> Artificial sequence

<400> 10

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420

gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt 480

tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg 540

gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac 600

agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc 660

ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct 720

tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg 780

aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg 840

aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc 900

gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc 960

aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc 1020

gcaacacctg aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc 1080

ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc 1140

agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc 1200

ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc 1260

agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac 1320

gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 1380

gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac 1440

cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1500

gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc 1560

ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg 1620

gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc 1680

aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag 1740

ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg 1800

acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag 1860

gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac 1920

ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg 1980

aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac 2040

caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa 2100

gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc 2160

caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa 2220

ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc 2280

agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat 2340

ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc 2400

ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag 2460

agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc 2520

cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg 2580

cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa 2640

tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc 2700

gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2760

ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac 2820

gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2880

aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac 2940

agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg 3000

aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac 3060

ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc 3120

gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc 3180

cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc 3240

ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg 3300

aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag 3360

accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc 3420

aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3480

gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg 3540

gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag 3600

gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac 3660

aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac 3720

gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg 3780

agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca 3840

aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag 3900

ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag 3960

gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac 4020

ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc 4080

gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa 4140

atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc 4200

gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa 4260

accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc 4320

atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 4380

tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct 4440

aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa 4500

gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc 4560

atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa 4620

gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac 4680

ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg 4740

ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc 4800

cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag 4860

atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac 4920

aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4980

atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac 5040

accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc 5100

caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac 5160

tctggcggct caaaaagaac cgccgacggc agcgaattca gcacagggag catgggaatg 5220

gactccctgg ccgagtctcg gtggcctccg ggcctggcag tcatgaagac aatagatgat 5280

ttgctgcggt gtggaatttg cttcgagtat ttcaacattg caatgataat acctcagtgt 5340

tcacataact actgctctct ctgtataaga aaatttctgt cctataaaac tcagtgtcca 5400

acttgctgtg tgactgtcac agagccggat ctgaaaaata accgcatatt agatgaactg 5460

gtaaaaagct tgaattttgc acggaatcat ctgctgcagt ttgctttaga gtcaccagcc 5520

aaatctcctg cttcttcctc ttcaaagaat cttgctgtca aagtatatac tcctgtagcc 5580

tccagacagt ctttaaagca ggggagcagg ttaatggata atttcttgat cagagaaatg 5640

agtggttcta catcagagtt gttgataaaa gaaaataaaa gcaaattcag ccctcaaaaa 5700

gaggcgagcc ctgctgcaaa gaccaaagag acacgttctg tagaagagat cgctccagat 5760

ccctcagagg ctaagcgtcc tgagccaccc tcgacatcca ctttgaaaca agttactaaa 5820

gtggattgtc ctgtttgcgg ggttaacatt ccagaaagtc acattaataa gcatttagac 5880

agctgtttat cacgcgaaga gaagaaggaa agcctcagaa gttctgttca caaaaggaag 5940

ccgcacatgt acaatgccca atgcgatgct ttgcatccta aatcagctgc tgaaatagtt 6000

cgagaaatcg aaaatataga gaagactagg atgcgtcttg aagctagtaa actcaatgaa 6060

agtgtaatgg tttttacaaa ggaccaaaca gaaaaggaaa tagatgaaat ccacagtaaa 6120

tatcgtaaaa aacataagag tgaatttcag cttctggtgg atcaggctag aaaaggatac 6180

aagaaaattg ctggaatgtc acaaaaaaca gtaacaataa caaaagaaga tgaatctaca 6240

gaaaagctat cttctgtatg catgggacag gaagataata tgacctcagt aacaaaccac 6300

ttttctcaat caaagctgga ctccccagag gaattggaac ctgacagaga agaggattct 6360

tctagctgta ttgatattca agaagttctt tcttcatcag aatcagattc atgcaatagt 6420

tccagttcag acatcataag agatctttta gaagaagagg aagcctggga agcatcacat 6480

aaaaacgatc ttcaagacac agaaataagt ccaagacaga atcgccgcac aagagccgct 6540

gaaagtgctg agattgaacc aagaaacaag cgtaatagga atgaaaaaag aaccgccgac 6600

ggcagcgagt tcgagcccaa gaagaagagg aaagtccaac cggtcatcat caccatcacc 6660

attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 6720

tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 6780

aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 6840

gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 6900

cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct 6960

ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 7020

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat 7080

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 7140

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 7200

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 7260

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 7320

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 7380

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 7440

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 7500

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 7560

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 7620

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 7680

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 7740

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 7800

ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 7860

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 7920

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 7980

atcctttgat cttttctacg gggtctgaca ctcagtggaa cgaaaactca cgttaaggga 8040

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 8100

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 8160

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 8220

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 8280

taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 8340

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 8400

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 8460

ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 8520

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 8580

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 8640

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 8700

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 8760

caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 8820

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 8880

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 8940

caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 9000

tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 9060

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 9120

cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt 9180

cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 9240

gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 9300

tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg 9360

tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta 9420

cggggtcatt agttcatagc ccatatattg agttccgcgt tacataactt acggtaaatg 9480

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 9540

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 9600

ctgcccactt ggcagtacat caagtgtatc 9630

<210> 11

<211> 8217

<212> DNA

<213> Artificial sequence

<400> 11

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420

gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt 480

tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg 540

gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac 600

agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc 660

ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct 720

tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg 780

aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg 840

aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc 900

gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc 960

aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc 1020

gcaacacctg aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc 1080

ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc 1140

agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc 1200

ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc 1260

agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac 1320

gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 1380

gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac 1440

cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1500

gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc 1560

ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg 1620

gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc 1680

aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag 1740

ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg 1800

acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag 1860

gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac 1920

ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg 1980

aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac 2040

caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa 2100

gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc 2160

caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa 2220

ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc 2280

agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat 2340

ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc 2400

ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag 2460

agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc 2520

cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg 2580

cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa 2640

tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc 2700

gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2760

ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac 2820

gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2880

aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac 2940

agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg 3000

aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac 3060

ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc 3120

gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc 3180

cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc 3240

ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg 3300

aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag 3360

accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc 3420

aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3480

gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg 3540

gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag 3600

gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac 3660

aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac 3720

gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg 3780

agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca 3840

aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag 3900

ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag 3960

gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac 4020

ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc 4080

gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa 4140

atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc 4200

gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa 4260

accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc 4320

atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 4380

tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct 4440

aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa 4500

gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc 4560

atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa 4620

gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac 4680

ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg 4740

ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc 4800

cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag 4860

atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac 4920

aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4980

atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac 5040

accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc 5100

caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac 5160

tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa 5220

gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5280

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 5340

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 5400

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 5460

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 5520

ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 5580

tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 5640

taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 5700

cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 5760

gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 5820

tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 5880

tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 5940

ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6000

agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6060

accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6120

ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6180

gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6240

ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6300

gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6360

taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 6420

tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6480

gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 6540

cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc 6600

agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 6660

cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 6720

cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 6780

ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 6840

taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 6900

tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 6960

ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7020

atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7080

gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7140

tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7200

cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7260

taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7320

ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 7380

ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 7440

cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 7500

ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 7560

gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 7620

gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 7680

aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 7740

gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 7800

agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 7860

aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 7920

ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 7980

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt 8040

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8100

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8160

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc 8217

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<400> 12

gaatactaag catagactcc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

ggagtctatg cttagtattc 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

gtaaacaaag catagactga 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

tcagtctatg ctttgtttac 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

gaacacaaag catagactgc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

gcagtctatg ctttgtgttc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

gatgagataa tgatgagtca 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> 19

tgactcatca ttatctcatc 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

gacaaaccag aagccgctcc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<400> 21

ggagcggctt ctggtttgtc 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

gggaataaat catagaatcc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

ggattctatg atttattccc 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

ggaacacaaa gcatagactg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

cagtctatgc tttgtgttcc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

gcacctacct cgggagctga 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<400> 27

tcagctcccg aggtaggtgc 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<400> 28

ggaatccctt ctgcagcacc 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<400> 29

ggtgctgcag aagggattcc 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<400> 30

tcagaaagtg gtggctggtg 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

caccagccac cactttctga 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<400> 32

ggcccagact gagcacgtga 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<400> 33

tcacgtgctc agtctgggcc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<400> 34

atatttgcat tgagatagtg 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<400> 35

cactatctca atgcaaatat 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<400> 36

gtcatcttag tcattacctg 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<400> 37

caggtaatga ctaagatgac 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<400> 38

gaagatagag aatagactgc 20

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence

<400> 39

gcagtctatt ctctatcttc 20

<210> 40

<211> 1791

<212> PRT

<213> Artificial sequence

<400> 40

Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile

325 330 335

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

340 345 350

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

355 360 365

Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1410 1415 1420

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

1425 1430 1435 1440

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

1445 1450 1455

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

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515 1520

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

1525 1530 1535

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

1540 1545 1550

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

1555 1560 1565

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

1570 1575 1580

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

1585 1590 1595 1600

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

1605 1610 1615

Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser

1620 1625 1630

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

1635 1640 1645

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

1650 1655 1660

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

1665 1670 1675 1680

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

1685 1690 1695

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

1700 1705 1710

His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys Tyr

1715 1720 1725

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

1730 1735 1740

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

1745 1750 1755 1760

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

1765 1770 1775

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

1780 1785 1790

<210> 41

<211> 1605

<212> PRT

<213> Artificial sequence

<400> 41

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

1 5 10 15

Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met

100 105 110

Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly

180 185 190

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

195 200 205

Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly

210 215 220

Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu

225 230 235 240

Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg

245 250 255

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

260 265 270

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

275 280 285

Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn

290 295 300

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

305 310 315 320

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

325 330 335

Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr

340 345 350

His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg

355 360 365

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

370 375 380

Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His

530 535 540

His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro

545 550 555 560

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

565 570 575

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

580 585 590

Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys

595 600 605

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

610 615 620

Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg

625 630 635 640

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

645 650 655

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

660 665 670

Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr

675 680 685

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

690 695 700

Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu

785 790 795 800

Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile

805 810 815

Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu

820 825 830

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

835 840 845

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

850 855 860

Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg

865 870 875 880

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

885 890 895

Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu

900 905 910

Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln

915 920 925

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

930 935 940

Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val

945 950 955 960

Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp

1140 1145 1150

Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

1220 1225 1230

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

1235 1240 1245

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

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

1445 1450 1455

His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu

1505 1510 1515 1520

Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala

1525 1530 1535

Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr

1540 1545 1550

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

1555 1560 1565

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

1570 1575 1580

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

1585 1590 1595 1600

Lys Lys Arg Lys Val

1605

Claims (13)

1. A fusion protein, characterized in that: the fusion protein comprises a first region and a second region from N-terminal to C-terminal, wherein the first region comprises ABEs comprising adenine deaminase or an enzymatically active component thereof and nCas 9; the second region comprises e18 protein; the fusion protein optionally comprises one or more linker amino acid sequences located in the first region and between the first region and the second region of the fusion protein.

2. The fusion protein of claim 1, wherein: the ABEs are one of ABE7.10, ABEmax and ABE8e base editors.

3. The fusion protein of claim 1, wherein: the fusion protein also includes a nuclear localization signal fragment.

4. The fusion protein of claim 1, wherein: the amino acid sequence of the fusion protein is shown in SEQ ID NO.40 when the ABEs is ABEmax; and when the ABEs are ABE8e, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 41.

5. An isolated polynucleotide, characterized in that: encoding the fusion protein of any one of claims 1 to 4.

6. The isolated polynucleotide of claim 5, wherein: the polynucleotide sequence comprises a first region encoding the ABEs and a second region encoding the e18 protein, and optionally one or more linker amino acid sequences located in the first region and between the first and second regions of the fusion protein.

7. The isolated polynucleotide of claim 5, wherein when said ABEs is ABEmax, said polynucleotide sequence is constructed by:

the gene sequence of ABEmax before gene modification is shown in SEQ ID NO. 2; the upstream primer sequence F1 of the e18 gene fragment 1 capable of effectively amplifying the target gene is shown as SEQ ID NO.3, and the downstream primer sequence R1 is shown as SEQ ID NO. 4; the upstream primer sequence F2 of the e18 gene fragment 2 capable of effectively amplifying the target gene is shown as SEQ ID NO.5, and the downstream primer sequence R2 is shown as SEQ ID NO. 6; the upstream primer sequence F3 of the e18 fragment gene sequence with the enzyme cutting sites which can be effectively amplified is shown as SEQ ID NO.7, the downstream primer sequence R3 is shown as SEQ ID NO.8, and the e18 gene sequence with double enzyme cutting sites is shown as SEQ ID NO. 9; the sequence obtained by connecting the fragment after the restriction of the enzyme SEQ ID NO.9 with the fragment after the restriction of the enzyme SEQ ID NO.2 is the polynucleotide sequence shown in SEQ ID NO. 1.

8. The isolated polynucleotide of claim 1, wherein when said ABEs is ABE8e, said polynucleotide sequence set forth in SEQ ID No.10 is constructed as follows; the gene sequence of ABE8e before gene modification is shown in SEQ ID NO. 11; the e18 gene sequence with double restriction sites is shown as SEQ ID NO.9, and the sequence obtained by connecting the fragment SEQ ID NO.9 with the fragment obtained by restriction of the enzyme SEQ ID NO.11 is the polynucleotide sequence shown as SEQ ID NO. 10.

9. A construct, characterized by: the construct comprises a polynucleotide according to any one of claims 5 to 8.

10. An expression system, characterized by: the expression system comprises the construct of claim 9 or a polynucleotide of any one of claims 5 to 8 integrated into the genome.

11. A use, characterized by: use of the fusion protein according to any one of claims 1 to 4 and the polynucleotide according to any one of claims 5 to 8 and the construct according to claim 9 or the expression system according to claim 10 for gene editing of converting the base A into G.

12. A base editing system characterized by: comprising the fusion protein of any one of claims 1 to 4 and sgRNAs.

13. A gene editing method characterized by comprising: comprising a fusion protein according to any one of claims 1 to 4 or a base editing system according to claim 12 for gene editing, wherein the gene editing is the conversion of the base A to G.

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