CN114763556B - Guide base editing system with improved gene editing efficiency and application thereof - Google Patents

Guide base editing system with improved gene editing efficiency and application thereof Download PDF

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CN114763556B
CN114763556B CN202011621689.1A CN202011621689A CN114763556B CN 114763556 B CN114763556 B CN 114763556B CN 202011621689 A CN202011621689 A CN 202011621689A CN 114763556 B CN114763556 B CN 114763556B
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CN114763556A (en
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徐雯
杨进孝
杨永星
康桂婷
李璐
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Beijing Academy of Agriculture and Forestry Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

The invention discloses a guiding base editing system with improved gene editing efficiency and application thereof. The guided base editing system comprises a fusion protein or a biological material associated with the fusion protein, pegRNA, or a biological material associated with the pegRNA; the fusion proteins include reverse transcriptase, cas9 nicking enzyme, self-cleaving oligopeptides, and selectable marker proteins; the pegRNA includes esgRNA, a reverse transcription template sequence, and a primer binding site sequence; the mutation bases introduced into the reverse transcription template sequence include a mutation base introduced at a target mutation site and an additional mutation base introduced at a site other than the target mutation site. Experiments prove that: the guiding base editing system can obviously improve the editing efficiency of the PE-P2 or PE-P3 guiding editing system.

Description

Guide base editing system with improved gene editing efficiency and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a guided base editing system with improved gene editing efficiency and application thereof.
Background
CRISPR-Cas9 technology has become a powerful means of genome editing and is widely used in many tissues and cells. The CRISPR/Cas9 protein-RNA complex is targeted by guide RNA (guide RNA), and cleavage produces a DNA double strand break (dsDNA break, DSB) that the organism then instinctively initiates a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ) and the other is homologous recombination (HDR). NHEJ is typically the majority, so repair produces random indels (insertions or deletions) that is much higher than exact repair. For precise base substitution, the use of HDR to achieve precise base substitution is greatly limited because of its inefficiency and the need for DNA templates. Cytosine base editors and adenine base editors reported successively in 2016 and 2017 can accurately realize transition from cytosine (Cytosine, C) to thymine (Thymine, T) and adenine (Adenine, a) to guanine (Guanine, G) without generating DSBs and without introducing DNA templates, but cannot realize transversion between purine and pyrimidine, i.e., a to T substitution, T to a substitution, C to G substitution, G to C substitution, a to C substitution, T to G substitution, C to a substitution, G to T substitution. Meanwhile, the base editor can only edit C or A in the active window, and when a plurality of C or A exist in the active window, the target C or A is easy to edit together with the non-target C or A, and the expected editing product cannot be finally obtained. All these drawbacks greatly limit the practical use of base editors.
In 2019, david Liu laboratories reported a new genome editing technology, i.e., guided editing technology (PRIME EDITING), and developed three kinds of guided editors (PE), PE1, PE2, and PE3, respectively. All three PEs were reverse transcriptase (REVERSE TRANSCRIPTASE, RT) fused together with Cas 9H 840A nickase (Cas 9n (H840A)) and genome editing was achieved using guide RNA (PRIME EDITING guide RNA, pegRNA). pegRNA in addition to the usual guide RNA (sgRNA), contain a RT template containing the base mutation of interest and a primer binding site (primer binding site, PBS). Experiments show that the technology can realize the editing of all 12 base substitution types in animal cell genome, breaks the limit of the traditional base editor and greatly improves the base editing range. Currently, in plants, although guided editing techniques can achieve all types of base substitution, there is still a problem in that base editing efficiency is not high or part of sites cannot be edited.
Disclosure of Invention
In a first aspect, the present invention protects a kit.
The complete system protected by the invention is a complete system A or a complete system B;
The kit of parts a comprises a reverse transcriptase or a biological material related thereto, a Cas9 nicking enzyme or a biological material related thereto, a selectable marker protein or a biological material related thereto, pegRNA or a biological material related to the pegRNA;
The set of systems b includes a fusion protein or a biological material associated with the fusion protein, pegRNA or a biological material associated with the pegRNA; the fusion proteins include reverse transcriptase, cas9 nicking enzyme, self-cleaving oligopeptides, and selectable marker proteins;
the pegRNA includes esgRNA, a reverse transcription template sequence (RT sequence), and a primer binding site sequence (PBS sequence); the mutation bases introduced into the reverse transcription template sequence include a mutation base introduced at a target mutation site and an additional mutation base introduced at a site other than the target mutation site.
In the above-mentioned complete set system, in the complete set system b, the fusion protein is any one of the following: fusion proteins consisting of Cas9 nicking enzyme, reverse transcriptase, self-cleaving oligopeptide and screening marker protein in sequence, fusion proteins consisting of screening marker protein, self-cleaving oligopeptide, cas9 nicking enzyme and reverse transcriptase in sequence, fusion proteins consisting of reverse transcriptase, cas9 nicking enzyme, self-cleaving oligopeptide and screening marker protein in sequence, and fusion proteins consisting of screening marker protein, self-cleaving oligopeptide, reverse transcriptase and Cas9 nicking enzyme in sequence.
In the above-described kit, the Cas9 nickase may be Cas9n (H840A);
The Cas9 nickase (Cas 9n (H840A)) may be various Cas9n or variants thereof known in the art, including bacterial-derived Cas9n (e.g., spCas9n, saCas9n-KKH, etc.), spCas9 variant nickases (e.g., xCas n, cas9n-NG, cas9n-VQR, cas9n-VRER, etc.) that recognize different PAMs, cas9 high-fidelity enzyme variant nickases (e.g., hypaCas n, eSpCas9 (1.1) n, cas9-HF1n, etc.), and the like.
Further, the Cas9n (H840A) is A1) or A2):
A1 Amino acid sequence is a protein shown in sequence 2;
a2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2.
Still further, the coding gene of Cas9n (H840A) is a 1) or a 2) or a 3):
a1 A cDNA molecule or a DNA molecule shown in positions 2293-6393 of sequence 1;
a2 A cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in a 1) and encoding the Cas9n (H840A);
a3 A cDNA molecule or a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in a 1) or a 2) and encodes the Cas9n (H840A).
In the above-described system set, the reverse transcriptase may be a reverse transcriptase derived from a virus, such as a reverse transcriptase derived from Moloney mouse leukemia virus (Moloney murine leukemia virus, M-MLV), a reverse transcriptase derived from cauliflower mosaic virus (CaMV), or the like, or may be a reverse transcriptase derived from a virus in bacteria, such as a reverse transcriptase derived from E.coli, or the like.
Further, the reverse transcriptase is a reverse transcriptase (M-MLV RT) derived from Moloney mouse leukemia virus; the M-MLV RT is B1) or B2):
b1 Amino acid sequence is a protein shown in sequence 3;
B2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3.
Still further, the coding gene of the M-MLV RT is b 1) or b 2) or b 3):
b1 A cDNA molecule or a DNA molecule shown in positions 6493-8523 of sequence 1;
b2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 1) and encoding said M-MLV RT;
b3 Under stringent conditions with the nucleotide sequence defined under b 1) or b 2) and a cDNA molecule or DNA molecule encoding said M-MLV RT.
In the above-described kit, the screening agent resistance protein is hygromycin phosphotransferase.
Further, the hygromycin phosphotransferase is D1) or D2):
D1 Amino acid sequence is a protein shown in sequence 4;
d2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4.
Still further, the coding gene of hygromycin phosphotransferase is d 1) or d 2) or d 3):
d1 A cDNA molecule or a DNA molecule shown at positions 8731-9756 of SEQ ID NO. 1;
d2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in d 1) and encoding said hygromycin phosphotransferase;
d3 Under stringent conditions with the nucleotide sequence defined in d 1) or d 2), and a cDNA molecule or DNA molecule encoding said hygromycin phosphotransferase.
In the above-described kit, the self-cleaving oligopeptide may be a 2A self-cleaving oligopeptide derived from a viral genome, such as a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine a rhinitis virus (ERAV) (E2A) peptide, a colleague-vein-lid-worm beta tetrad virus (Thosea asigna virus) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a taylor virus 2A peptide, and an encephalomyocarditis virus 2A peptide.
Further, the self-cleaving oligopeptide is a 2A self-cleaving oligopeptide (P2A) derived from porcine teschovirus-1; the amino acid sequence of P2A is C1) or C2):
c1 Amino acid sequence is a protein shown in sequence 5;
C2 A protein having the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 5.
Still further, the coding gene of P2A is c 1) or c 2) or c 3):
c1 A cDNA molecule or a DNA molecule shown at positions 8674-8730 of SEQ ID NO. 1;
c2 A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in c 1) and encoding said P2A;
c3 Under stringent conditions with the nucleotide sequence defined under c 1) or c 2), and a cDNA molecule or DNA molecule encoding said P2A.
In the complete system, pegRNA consists of a target sequence (marked as target sequence A), a esgRNA framework, an RT sequence and a PBS sequence in sequence.
The RT sequence is the reverse complementary sequence of 3 bases at the 3' -end of the target sequence and a section of genome sequence which is continuous behind the target sequence, and target mutation is introduced into the RT sequence, and is used as a reverse transcription template of reverse transcriptase, cDNA is reversely transcribed, and then the RT sequence is used as a repair template to repair genome DNA. The RT sequence can further be 8-34bp in size.
Typically, the RT sequence contains only the target mutation site (i.e., the site where mutation is desired) and the mutation base is introduced at the target mutation site, but the invention introduces the mutation base at a site other than the target mutation site (denoted as an additional mutation site) in addition to the mutation base at the target mutation site. The additional mutation site may be any site in the RT sequence other than the mutation site of interest. As shown in FIG. 2, taking the ALS-1 target as an example, the RT design form (RT-S template form) only comprises the target mutation site, i.e. the mutation base T is introduced at the target mutation site (+1) in the RT sequence, while the RT-M template form comprises other mutation sites besides the target mutation site, i.e. the mutation base A is introduced at the additional mutation sites (+2 and +5) in the RT sequence in addition to the mutation base T introduced at the target mutation site (+1). In practical application, a proper site can be selected as an additional mutation site according to practical needs, and a proper mutation base is introduced, for example, when only the base of the target mutation site is expected to be mutated to cause the change of the amino acid sequence, and the base of the additional mutation site is not caused to cause the change of the amino acid sequence after the mutation, synonymous mutation can be carried out on the base of the additional mutation site through design.
Further, the way of introducing the mutant base is base substitution.
Further, the number of mutant bases introduced at the target mutation site may be one or two or more. In an embodiment of the present invention, the number of mutation bases introduced at the target mutation site is specifically one.
The number of additional mutation bases introduced at other sites than the target mutation site may be one or two or more. In embodiments of the invention, the number of additional mutation bases introduced at other sites than the target mutation site is specifically two or three.
The PBS sequence (primer binding site sequence) is the reverse complementary sequence (n <17 > which is 1-n) of the target sequence from the nth base to the 17 th base of the 5' end of the target sequence.
The design methods or principles of the RT sequences and the PBS sequences can be referred to those reported in the prior art as related to the design methods or principles of the RT sequences and the PBS sequences of pegRNA in the guided editing technique (PRIME EDITING, PE).
The esgRNA skeleton is F1) or F2) or F3):
f1 An RNA molecule obtained by replacing T in positions 11008 to 11093 of the sequence 1 with U;
F2 An RNA molecule having the same function and obtained by substituting and/or deleting and/or adding one or more nucleotides to the RNA molecule shown in F1);
F3 An RNA molecule having 75% or more identity and the same function as the nucleotide sequence defined in F1) or F2).
The kit may also include esgRNA or biological materials associated with esgRNA.
The esgRNA consists of a target sequence (marked as target sequence B) and the esgRNA framework. This esgRNA is used to create a non-editing chain cut, the site of which can be chosen arbitrarily. The target sequence A and the target sequence B are respectively positioned on two chains of the target DNA, and the target sequence A and the target sequence B can be in complementary coincidence or partial complementary coincidence, or can have a certain distance.
The application of the complete system is as follows:
s1) editing genome sequences of organisms or biological cells;
s2) preparing an edited product of genomic sequences of an organism or a biological cell;
S3) improving the editing efficiency of genome sequences of organisms or biological cells;
S4) preparing a product for improving the editing efficiency of genome sequences of organisms or biological cells.
In a second aspect, the present invention provides a novel use of the above-described kit or the above pegRNA.
The invention protects the use of the above-described kit or of the above pegRNA in any one of the following S1) to S4):
s1) editing genome sequences of organisms or biological cells;
s2) preparing an edited product of genomic sequences of an organism or a biological cell;
S3) improving the editing efficiency of genome sequences of organisms or biological cells;
S4) preparing a product for improving the editing efficiency of genome sequences of organisms or biological cells.
In a third aspect, the invention protects a method as described in any one of T1) to T5) below:
T1) a method for editing a genomic sequence or a method for improving the efficiency of editing a genomic sequence of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the reverse transcriptase, the Cas9 nicking enzyme, the selectable marker protein, and the pegRNA; the pegRNA targets a target sequence A and is used for editing a genome sequence.
T2) a method for editing a genomic sequence or a method for improving the efficiency of editing a genomic sequence of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the reverse transcriptase, the Cas9 nicking enzyme, the selectable marker protein, the pegRNA, and the esgRNA; the pegRNA is used for targeting a target sequence A and editing a genome sequence; and esgRNA is used for targeting the target sequence B and generating a notch on a non-editing chain so as to improve the editing efficiency of the target mutation.
T3) a method for editing a genomic sequence or a method for improving the efficiency of editing a genomic sequence of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the fusion protein and pegRNA; the pegRNA targets a target sequence A and is used for editing a genome sequence.
T4) a method of editing a genomic sequence or a method of improving the efficiency of editing a genomic sequence of an organism or a biological cell, comprising the steps of: allowing an organism or biological cell to express the fusion protein, the pegRNA, and the esgRNA; the pegRNA is used for targeting a target sequence A and editing a genome sequence; and esgRNA is used for targeting the target sequence B and generating a notch on a non-editing chain so as to improve the editing efficiency of the target mutation.
T5) a method for preparing a biological mutant, comprising the following steps: editing the genome sequence of the organism or the biological cell according to the method described in T1) -T4) to obtain the biological mutant.
In the above method, in T1), the step of allowing the organism or the biological cell to express the reverse transcriptase, the Cas9 nicking enzyme, the selectable marker protein, and pegRNA is a step of introducing a gene encoding the reverse transcriptase, a gene encoding the Cas9 nicking enzyme, a gene encoding the selectable marker protein, and a DNA molecule transcribed from pegRNA into a plant of interest.
In T2), the expression of the reverse transcriptase, the Cas9 nicking enzyme, the selectable marker protein, the pegRNA, and the esgRNA by the organism or biological cell is performed by introducing a gene encoding the reverse transcriptase, a gene encoding the Cas9 nicking enzyme, a gene encoding the selectable marker protein, a DNA molecule that transcribes the pegRNA, and a DNA molecule that transcribes the esgRNA into a plant of interest.
In T3), the method for expressing the fusion protein and pegRNA in an organism or a biological cell is to introduce the gene encoding the fusion protein and a DNA molecule transcribed from pegRNA into a plant of interest.
In the T4), the method for expressing the fusion protein, pegRNA and esgRNA in an organism or a biological cell is to introduce the gene encoding the fusion protein, the DNA molecule transcribed from pegRNA and the DNA molecule transcribed from esgRNA into a plant of interest.
Further, in the T4), the gene encoding the fusion protein, the DNA molecule transcribed from pegRNA and the DNA molecule transcribed from esgRNA are introduced into a plant of interest through a recombinant expression vector. The coding gene of the fusion protein, the DNA molecule transcribed into pegRNA and the DNA molecule transcribed into esgRNA can be introduced into a target plant through the same recombinant expression vector or can be commonly introduced into the target plant through two or more recombinant expression vectors.
Furthermore, the encoding gene of the fusion protein, the DNA molecule transcribed into pegRNA and the DNA molecule transcribed into esgRNA are introduced into the target plant through the same recombinant expression vector. In one embodiment of the invention, the recombinant expression vector comprises an expression cassette consisting of a promoter, a coding gene of Cas9n (H840A), a coding gene of reverse transcriptase M-MLV RT, a coding gene of self-cleaving oligopeptide P2A, a coding gene of screening agent resistance protein HPT and a terminator in sequence, an expression cassette consisting of a promoter, a DNA molecule of the transcript esgRNA and a poly T in sequence, and an expression cassette consisting of a promoter, a DNA molecule of the transcript pegRNA and a poly T in sequence. In another embodiment of the present invention, the recombinant expression vector comprises an expression cassette consisting of a promoter, a gene encoding reverse transcriptase M-MLV RT, a gene encoding Cas9n (H840A), a gene encoding self-cleaving oligopeptide P2A, a gene encoding a screening agent resistance protein HPT, and a terminator in this order, an expression cassette consisting of a promoter, a DNA molecule of the transcript esgRNA, and a poly T in this order, and an expression cassette consisting of a promoter, a DNA molecule of the transcript pegRNA, and a poly T in this order.
In any of the above kits, or applications or methods, the editing of the genomic sequence is a base substitution of the genomic sequence.
In any of the above kits or uses or methods, the organism is X1) or X2) or X3) or X4):
X1) plants or animals;
X2) monocotyledonous or dicotyledonous plants;
x3) a gramineous plant;
X4) rice.
The biological cell is Y1) or Y2) or Y3) or Y4):
y1) plant cells or animal cells;
Y2) monocot or dicot cells;
y3) a graminaceous plant cell;
Y4) rice cells.
The invention improves the reverse transcription template sequence in the guide editing system, introduces a mutation base at a target mutation site in the reverse transcription template sequence, and introduces an additional mutation base at other sites except the target mutation site. Experiments prove that: the improved guide editing system can obviously improve the editing efficiency of the PE-P2 or PE-P3 guide editing system.
Drawings
FIG. 1 is a schematic diagram showing the structures of expression vectors of the guidance editing system PE-P3 and the guidance editing system PE-P2.
FIG. 2 is a schematic representation of the RT-M template form and the RT-S template form.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
The names and sequences of the primers used for amplifying the target gene in the following examples are shown in the following table.
In the following examples of the present invention,
Guide editor callus edit efficiency = (number of reads with all mutation sites detected in group 1/total number of reads x 100% + number of reads with all mutation sites detected in group 2/total number of reads x 100% + number of reads with all mutation sites detected in group 3/total number of reads x 100%)/3.
Guidance editor T0 shoot edit efficiency = number of positive T0 shoots mutated at all mutation sites/total positive T0 shoots analyzed x 100%.
Paddy rice in Nippon sunny days: reference is made to: liang Weigong, wang Gaohua, du Jingyao, et al sodium nitroprusside and its photolysis products have an effect on the growth of young seedlings of Nippon rice and the expression of 5 hormone marker genes [ J ]. University of Henan university (Nature edition), 2017 (2): 48-52; the public is available from the academy of agriculture and forestry, beijing, city.
Recovery medium: n6 solid medium containing 200mg/L of timentin.
Screening the culture medium: n6 solid medium containing 50mg/L hygromycin.
Differentiation medium: n6 solid medium containing 2mg/L KT, 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.
Rooting medium: n6 solid medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.
Embodiment 1, method for designing different guidance editing System
The guided editing system includes fusion proteins, esgRNA, and pegRNA; fusion proteins include Cas9 nicking enzymes (e.g., cas9n (H840A)), reverse transcriptases (e.g., M-MLV), self-cleaving oligopeptides (e.g., P2A), and selectable marker proteins (e.g., HPT); pegRNA consists of esgRNA, a reverse transcription template sequence (RT sequence) and a primer binding site sequence (PBS sequence) in this order.
1. Design for reverse transcriptase and Cas9 nicking enzyme in guided editing system
According to the difference of the connection modes of reverse transcriptase and Cas9 nickase, two guiding editing systems are divided: the guiding editing system PE-P2 in the prior art and the guiding editing system PE-P3 designed by the invention. The schematic structures of the expression vectors of the guidance editing system PE-P3 and the guidance editing system PE-P2 are shown in FIG. 1.
Expression vectors guiding the editing system PE-P2 include Cas9n (H840A) & M-MLV & Hpt expression cassettes, esgRNA expression cassettes, and pegRNA expression cassettes. In the Cas9n (H840A) and M-MLV & Hpt expression cassette, M-MLV is fused at the C end of Cas9n (H840A) and fused with the screening agent resistance protein through self-cleaving polypeptide P2A.
Expression vectors guiding the editing system PE-P3 include M-MLV & Cas9n (H840A) & Hpt expression cassettes, esgRNA expression cassettes and pegRNA expression cassettes. In the M-MLV & Cas9N (H840A) and Hpt expression cassette, M-MLV is fused at the N end of Cas9N (H840A) and fused with the screening agent resistance protein through self-cleaving polypeptide P2A.
2. Design for reverse transcription template in guided editing system
Based on the guidance editing system PE-P2 and the guidance editing system PE-P3, the reverse transcription templates (RT templates) are divided into two forms according to whether additional mutation bases are introduced into the reverse transcription templates: an RT-S template form and an RT-M template form. Taking the target in FIG. 2 as an example, a schematic diagram of the RT-M template form and the RT-S template form is shown in FIG. 2.
RT-S template form: compared with the genome sequence, the RT template only contains a single mutation base, the single mutation base site is marked as a target mutation site, namely, in the RT-S template form, the mutation base is only introduced into the target mutation site, and all mutation sites are only target mutation sites.
RT-M template form: with respect to the genomic sequence, an additional mutation base is introduced into the RT template on the basis of RT-S, and the additional mutation base site is designated as an additional mutation site, namely, in the form of the RT-M template, the additional mutation base is introduced into other sites (additional mutation sites) except the target mutation site, and all mutation sites consist of the target mutation site and the additional mutation site.
Example 2 construction of expression vectors of different guide editing systems and comparison of efficiency of base editing of Rice genome
1. Construction of expression vectors for different guided editing systems
The following recombinant vectors were artificially synthesized, each of which was a circular plasmid:
The total number of expression vectors of the guide editing system PE-P2 is 14, and PE-P2-1,PE-P2-2,PE-P2-3,PE-P2-4,PE-P2-5,PE-P2-6,PE-P2-7,PE-P2-8,PE-P2-9,PE-P2-10,PE-P2-11,PE-P2-12,PE-P2-13,PE-P2-14 vectors are respectively used.
The total number of expression vectors of the guide editing system PE-P3 is 14, and PE-P3-1,PE-P3-2,PE-P3-3,PE-P3-4,PE-P3-5,PE-P3-6,PE-P3-7,PE-P3-8,PE-P3-9,PE-P3-10,PE-P3-11,PE-P3-12,PE-P3-13,PE-P3-14 vectors are respectively used.
The sequence of the PE-P2-1 recombinant expression vector is sequence 1 in a sequence table. Wherein, the 102-2073 position of the sequence 1 is the nucleotide sequence of ZmUbi1 promoter, the 2293-6393 is the coding sequence (without termination codon) of Cas9n (H840A) protein, and the coding sequence 2 is shown as Cas9n (H840A) protein; the 6493-8523 position of the sequence 1 is the coding sequence of the M-MLV RT protein, and the coding sequence 3 shows the M-MLV RT protein; the 8674-8730 of the sequence 1 is the coding sequence of P2A, and the coding sequence 5 shows the protein; positions 8731-9756 of the sequence 1 are coding sequences of hygromycin phosphotransferase, and the coding sequences are hygromycin phosphotransferase protein shown in the sequence 4; positions 9763-10017 of sequence 1 are the Nos terminator sequence; nucleotide sequence of OsU a promoter at 10026-10491 position, esgRNA target sequence for generating non-coding strand incision at 10492-10511 position, esgRNA skeleton sequence for generating non-coding strand incision at 10512-10597 position, and Poly T at 10598-10606 position in sequence 1; nucleotide sequence of OsU promoter at 10607-10987, pegRNA-01 target sequence at 10988-11007, esgRNA skeleton sequence corresponding to pegRNA-01 at 11008-11093, RT & PBS sequence on pegRNA-01 at 11094-11120, and Poly T at 11121-11128 in sequence 1; the sequence of esgRNA target, pegRNA-01 target and RT & PBS sequence on pegRNA-01 in PE-P2-1 recombinant expression vector for generating non-coding strand incision is shown in Table 1.
The sequence of the PE-P2-2 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-02 target sequence and the RT & PBS sequence on pegRNA-02, which generate the non-coding chain notch, which correspond to pegRNA-02, respectively, and keeping other sequences unchanged. pegRNA-02 corresponds to the esgRNA target sequence, pegRNA-02 target sequence and pegRNA-02 RT & PBS sequence for the generation of non-coding strand breaks as shown in Table 1.
The sequence of the PE-P2-3 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-03 target sequence and the RT & PBS sequence on pegRNA-03, which generate the non-coding chain notch, which correspond to pegRNA-03, respectively, and keeping other sequences unchanged. pegRNA-03 corresponds to the esgRNA target sequence, pegRNA-03 target sequence and pegRNA-03 RT & PBS sequence for the non-coding strand breaks shown in Table 1.
The sequence of the PE-P2-4 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-04 target sequence and RT & PBS sequence on pegRNA-04, which generate non-coding chain cuts, corresponding to pegRNA-04, respectively, and keeping other sequences unchanged. pegRNA-04, the corresponding esgRNA target sequence for non-coding strand breaks, the pegRNA-04 target sequence and the RT & PBS sequence on pegRNA-04 are shown in Table 1.
The sequence of the PE-P2-5 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-05 target sequence and RT & PBS sequence on pegRNA-05, which generate non-coding chain cuts, corresponding to pegRNA-05, respectively, and keeping other sequences unchanged. pegRNA-05 corresponds to the esgRNA target sequence, pegRNA-05 target sequence and pegRNA-05 RT & PBS sequence for the non-coding strand nick.
The sequence of the PE-P2-6 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-06 target sequence and the RT & PBS sequence on pegRNA-06, which generate non-coding chain cuts, corresponding to pegRNA-06, respectively, and keeping other sequences unchanged. pegRNA-06 corresponds to the esgRNA target sequence, pegRNA-06 target sequence and pegRNA-06 RT & PBS sequence for generating non-coding strand breaks as shown in Table 1.
The sequence of the PE-P2-7 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-07 target sequence and the RT & PBS sequence on pegRNA-07, which generate non-coding chain cuts, corresponding to pegRNA-07, respectively, and keeping other sequences unchanged. pegRNA-07 corresponds to the esgRNA target sequence for generating a non-coding strand cut, pegRNA-07 target sequence and the RT & PBS sequence on pegRNA-07 are shown in Table 1.
The sequence of the PE-P2-8 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-08 target sequence and the RT & PBS sequence on pegRNA-08, which generate non-coding chain cuts, corresponding to pegRNA-08, respectively, and keeping other sequences unchanged. pegRNA-08, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-08 target sequence, and the RT & PBS sequence on pegRNA-08 are shown in Table 1.
The sequence of the PE-P2-9 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-09 target sequence and the RT & PBS sequence on pegRNA-09, which generate the non-coding chain notch, which correspond to pegRNA-09, respectively, and keeping other sequences unchanged. pegRNA-09, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-09 target sequence, and the RT & PBS sequence on pegRNA-09 are shown in Table 1.
The sequence of the PE-P2-10 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-10 target sequence and the RT & PBS sequence on pegRNA-10, which generate the non-coding chain notch, corresponding to pegRNA-10, respectively, and keeping other sequences unchanged. pegRNA-10 corresponds to the esgRNA target sequence for non-coding strand breaks, pegRNA-10 target sequence and the RT & PBS sequence on pegRNA-10 as shown in Table 1.
The sequence of the PE-P2-11 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-11 target sequence and the RT & PBS sequence on pegRNA-11, which generate the non-coding chain notch, which correspond to pegRNA-11, respectively, and keeping other sequences unchanged. pegRNA-11, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-11 target sequence, and the RT & PBS sequence on pegRNA-11 are shown in Table 1.
The sequence of the PE-P2-12 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-12 target sequence and the RT & PBS sequence on pegRNA-12, which generate the non-coding chain notch, which correspond to pegRNA-12, respectively, and keeping other sequences unchanged. pegRNA-12 corresponds to the esgRNA target sequence for generating a non-coding strand incision, pegRNA-12 target sequence and the RT & PBS sequence on pegRNA-12 are shown in Table 1.
The sequence of the PE-P2-13 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-13 target sequence and the RT & PBS sequence on pegRNA-13, which generate the non-coding chain notch, which correspond to pegRNA-13, respectively, and keeping other sequences unchanged. pegRNA-13, the corresponding esgRNA target sequence for non-coding strand breaks, the pegRNA-13 target sequence and the RT & PBS sequence on pegRNA-13 are shown in Table 1.
The sequence of the PE-P2-14 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P2-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-14 target sequence and the RT & PBS sequence on pegRNA-14, which generate non-coding chain cuts, corresponding to pegRNA-14, respectively, and keeping other sequences unchanged. pegRNA-14, the corresponding esgRNA target sequence for generating a non-coding strand incision, the pegRNA-14 target sequence, and the RT & PBS sequence on pegRNA-14 are shown in Table 1.
The sequence of the PE-P3-1 recombinant expression vector is a sequence 6 in a sequence table. Wherein, the 102-2073 position of the sequence 6 is the nucleotide sequence of ZmUbi1 promoter, the 2290-4320 position is the coding sequence of M-MLV RT protein, and the coding sequence is M-MLV RT protein shown in the sequence 3; the 4420-8520 of the sequence 6 is a coding sequence (without a stop codon) of the Cas9n (H840A) protein, and the coding sequence of the Cas9n (H840A) protein shown in the sequence 2; the 8671-8727 bits of the sequence 6 are the coding sequence of P2A, and the protein shown in the sequence 5 is coded; the 8728-9753 of the sequence 6 is the coding sequence of hygromycin phosphotransferase, and the coding sequence of hygromycin phosphotransferase protein shown in the sequence 4; positions 9760-10014 of sequence 6 are Nos terminator sequences; the nucleotide sequence of the OsU a promoter at the 10023-10488 position of the sequence 6, the esgRNA target sequence for generating a non-coding chain incision at the 10489-10508 position, the esgRNA framework sequence for generating a non-coding chain incision at the 10509-10594 position and the Poly T at the 10595-10603 position; nucleotide sequence of OsU promoter at position 10604-10984, pegRNA-01 target sequence at position 10985-11004, esgRNA skeleton sequence corresponding to pegRNA-01 at position 11005-11090, RT & PBS sequence on pegRNA-01 at position 11091-11117, and Poly T at position 11118-11125 of sequence 6; the sequence of esgRNA target, pegRNA-01 target and RT & PBS sequence on pegRNA-01 in PE-P3-1 recombinant expression vector for generating non-coding strand incision is shown in Table 1.
The sequence of the PE-P3-2 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-02 target sequence and RT & PBS sequence on pegRNA-02, which generate non-coding chain cuts, corresponding to pegRNA-02, respectively, and keeping other sequences unchanged. pegRNA-02 corresponds to the esgRNA target sequence, pegRNA-02 target sequence and pegRNA-02 RT & PBS sequence for the generation of non-coding strand breaks as shown in Table 1.
The sequence of the PE-P3-3 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-03 target sequence and the RT & PBS sequence on pegRNA-03, which generate the non-coding chain notch, which correspond to pegRNA-03, respectively, and keeping other sequences unchanged. pegRNA-03 corresponds to the esgRNA target sequence, pegRNA-03 target sequence and pegRNA-03 RT & PBS sequence for the non-coding strand breaks shown in Table 1.
The sequence of the PE-P3-4 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-04 target sequence and RT & PBS sequence on pegRNA-04, which generate non-coding chain cuts, corresponding to pegRNA-04, respectively, and keeping other sequences unchanged. pegRNA-04, the corresponding esgRNA target sequence for non-coding strand breaks, the pegRNA-04 target sequence and the RT & PBS sequence on pegRNA-04 are shown in Table 1.
The sequence of the PE-P3-5 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-05 target sequence and RT & PBS sequence on pegRNA-05, which generate non-coding chain cuts, corresponding to pegRNA-05, respectively, and keeping other sequences unchanged. pegRNA-05 corresponds to the esgRNA target sequence, pegRNA-05 target sequence and pegRNA-05 RT & PBS sequence for the non-coding strand nick.
The sequence of the PE-P3-6 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-06 target sequence and the RT & PBS sequence on pegRNA-06, which generate non-coding chain cuts, corresponding to pegRNA-06, respectively, and keeping other sequences unchanged. pegRNA-06 corresponds to the esgRNA target sequence, pegRNA-06 target sequence and pegRNA-06 RT & PBS sequence for generating non-coding strand breaks as shown in Table 1.
The sequence of the PE-P3-7 recombinant expression vector is obtained by replacing the esgRNA target sequence, pegRNA-01 target sequence and RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, pegRNA-07 target sequence and RT & PBS sequence on pegRNA-07, which generate non-coding chain cuts, corresponding to pegRNA-07, respectively, and keeping other sequences unchanged. pegRNA-07 corresponds to the esgRNA target sequence for generating a non-coding strand cut, pegRNA-07 target sequence and the RT & PBS sequence on pegRNA-07 are shown in Table 1.
The sequence of the PE-P3-8 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-08 target sequence and the RT & PBS sequence on pegRNA-08, which generate non-coding chain cuts, corresponding to pegRNA-08, respectively, and keeping other sequences unchanged. pegRNA-08, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-08 target sequence, and the RT & PBS sequence on pegRNA-08 are shown in Table 1.
The sequence of the PE-P3-9 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-09 target sequence and the RT & PBS sequence on pegRNA-09, which generate the non-coding chain notch, which correspond to pegRNA-09, respectively, and keeping other sequences unchanged. pegRNA-09, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-09 target sequence, and the RT & PBS sequence on pegRNA-09 are shown in Table 1.
The sequence of the PE-P3-10 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-10 target sequence and the RT & PBS sequence on pegRNA-10, which generate the non-coding chain notch, corresponding to pegRNA-10, respectively, and keeping other sequences unchanged. pegRNA-10 corresponds to the esgRNA target sequence for non-coding strand breaks, pegRNA-10 target sequence and the RT & PBS sequence on pegRNA-10 as shown in Table 1.
The sequence of the PE-P3-11 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-11 target sequence and the RT & PBS sequence on pegRNA-11, which generate the non-coding chain notch, which correspond to pegRNA-11, respectively, and keeping other sequences unchanged. pegRNA-11, the corresponding esgRNA target sequence for generating a non-coding strand cut, the pegRNA-11 target sequence, and the RT & PBS sequence on pegRNA-11 are shown in Table 1.
The sequence of the PE-P3-12 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-12 target sequence and the RT & PBS sequence on pegRNA-12, which generate the non-coding chain notch, which correspond to pegRNA-12, respectively, and keeping other sequences unchanged. pegRNA-12 corresponds to the esgRNA target sequence for generating a non-coding strand incision, pegRNA-12 target sequence and the RT & PBS sequence on pegRNA-12 are shown in Table 1.
The sequence of the PE-P3-13 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate the non-coding chain notch, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-13 target sequence and the RT & PBS sequence on pegRNA-13, which generate the non-coding chain notch, which correspond to pegRNA-13, respectively, and keeping other sequences unchanged. pegRNA-13, the corresponding esgRNA target sequence for non-coding strand breaks, the pegRNA-13 target sequence and the RT & PBS sequence on pegRNA-13 are shown in Table 1.
The sequence of the PE-P3-14 recombinant expression vector is obtained by replacing the esgRNA target sequence, the pegRNA-01 target sequence and the RT & PBS sequence on pegRNA-01, which generate non-coding chain cuts, in the PE-P3-1 recombinant expression vector sequence with the esgRNA target sequence, the pegRNA-14 target sequence and the RT & PBS sequence on pegRNA-14, which generate non-coding chain cuts, corresponding to pegRNA-14, respectively, and keeping other sequences unchanged. pegRNA-14, the corresponding esgRNA target sequence for generating a non-coding strand incision, the pegRNA-14 target sequence, and the RT & PBS sequence on pegRNA-14 are shown in Table 1.
The target nucleotide sequence and RT & PBS sequences on pegRNA of each vector and esgRNA target sequence for use in generating non-coding strand breaks are shown in table 1.
TABLE 1
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2. Rice resistant callus and acquisition of positive T0 seedlings
Carrying out operations on PE-P2-1,PE-P2-2,PE-P2-3,PE-P2-4,PE-P2-5,PE-P2-6,PE-P2-7,PE-P2-8,PE-P2-9,PE-P2-10,PE-P2-11,PE-P2-12,PE-P2-13,PE-P2-14,PE-P3-1,PE-P3-2,PE-P3-3,PE-P3-4,PE-P3-5,PE-P3-6,PE-P3-7,PE-P3-8,PE-P3-9,PE-P3-10,PE-P3-11,PE-P3-12,PE-P3-13 and PE-P3-14 recombinant expression vectors constructed in the first step according to the following steps 1-9 respectively:
1. The vector was introduced into Agrobacterium EHA105 (product of Shanghai Di Biotechnology Co., ltd.; CAT#: AC 1010) to obtain recombinant Agrobacterium.
2. Recombinant Agrobacterium was cultured using a medium (YEP medium containing 50. Mu.g/ml kanamycin and 25. Mu.g/ml rifampicin), shake-cultured at 28℃at 150rpm until OD 600 was 1.0-2.0, centrifuged at 10000rpm for 1min at room temperature, and the cells were resuspended with an invading solution (the sugar in the N6 liquid medium was replaced with glucose and sucrose at concentrations of 10g/L and 20g/L, respectively) and diluted to OD 600 of 0.2 to give an Agrobacterium invading solution.
3. Removing shells of mature seeds of a rice variety Japanese sunny day, putting the mature seeds into a 100mL triangular flask, adding 70% (v/v) ethanol aqueous solution for soaking for 30sec, putting the mature seeds into 25% (v/v) sodium hypochlorite aqueous solution, vibrating and sterilizing for 30min at 120rpm, washing with sterile water for 3 times, sucking water by using filter paper, putting seed embryos downwards on an N6 solid medium, and culturing in dark at 28 ℃ for 4-6 weeks to obtain rice calli.
4. After the step 3 is completed, the rice callus is soaked in agrobacterium infection solution A (the agrobacterium infection solution A is a liquid obtained by adding acetosyringone into the agrobacterium infection solution, the addition amount of the acetosyringone satisfies the volume ratio of the acetosyringone to the agrobacterium infection solution is 25 mu l:50 ml) for 10min, and then the rice callus is placed on a culture dish (containing about 200ml of infection solution without agrobacterium) paved with two layers of sterilization filter paper, and is subjected to dark culture at 21 ℃ for 1 day.
5. And (3) putting the rice callus obtained in the step (4) on a recovery culture medium, and carrying out dark culture at 25-28 ℃ for 3 days.
6. And (3) taking the rice callus obtained in the step (5), placing the rice callus on a screening culture medium, and culturing the rice callus in dark at 28 ℃ for 2 weeks.
7. And (3) taking the rice callus obtained in the step (6), and placing the rice callus on a screening culture medium again, and carrying out dark culture at 28 ℃ for 2 weeks to obtain the rice resistant callus.
8. And (3) placing the rice resistant callus obtained in the step (7) on a differentiation medium, culturing for about 1 month at 25 ℃ by illumination, transferring the differentiated plantlet onto a rooting medium, and culturing for 2 weeks at 25 ℃ by illumination to obtain the rice T0 plantlet.
9. Extracting genome DNA of the obtained rice T0 seedling as a template for PE-P2-1,PE-P2-2,PE-P2-3,PE-P2-4,PE-P2-5,PE-P2-6,PE-P2-7,PE-P2-8,PE-P2-9,PE-P2-10,PE-P2-11,PE-P2-12,PE-P2-13,PE-P2-14 recombinant expression vectors, and carrying out PCR amplification by using a primer pair consisting of a primer F (5'-GATCTTGATATACTTGGATGATGGC-3') and a primer R (5'-GGGGTACTTCTCGTGGTAGG-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 753bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 753bp, the corresponding rice T0 seedling is not a rice positive T0 seedling. Extracting genome DNA of the obtained rice T0 seedling as a template for PE-P3-1,PE-P3-2,PE-P3-3,PE-P3-4,PE-P3-5,PE-P3-6,PE-P3-7,PE-P3-8,PE-P3-9,PE-P3-10,PE-P3-11,PE-P3-12,PE-P3-13 and PE-P3-14 recombinant expression vectors, and carrying out PCR amplification by using a primer pair consisting of a primer F (5'-GATCTTGATATACTTGGATGATGGC-3') and a primer R (5'-ATGACTGTCTCCTTCCTTGCC-3') to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 1220bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 1220bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.
3. Analysis of results
1. And (3) randomly selecting 24 resistant calli obtained in the step (7) in the step (II) for each vector, extracting DNA, randomly mixing 8 calli DNA, and finally obtaining 3 mixed DNA, namely dividing into 3 groups. Taking the mixed DNA as a template, and carrying out PCR amplification on OsALS-1 targets by adopting a primer pair OsALS-1 to obtain a first round of PCR amplification product; for OsACC-2 targets, carrying out PCR amplification by adopting a primer pair OsACC-2 to obtain a first round PCR amplification product; for OsWaxy-1 targets, carrying out PCR amplification by adopting a primer pair OsWaxy-1 to obtain a first round PCR amplification product; for OsDEP1 targets, carrying out PCR amplification by adopting a primer pair OsDEP to obtain a first round PCR amplification product; for OsALS-2 targets, the primer pair OsALS-2 is adopted for PCR amplification to obtain the first round PCR amplification product. The first round of PCR products were used as templates, and different forward and reverse barcodes were added to the ends of the PCR products to construct libraries, forming mixed libraries, which were sequenced using Illumina NovaSeq high throughput sequencing platform, each mixed library sequencing data amount 2G (Beijing nozaku source technologies Co., ltd.). Sequencing results were analyzed only for each pegRNA region, leading to an editor callus editing efficiency of 3 sets of averages of the ratio of the number of reads detected with all mutation sites to the total number of reads. The experimental results are shown in Table 2.
The results show that for the mutation of target OsALS-1 (+1G/T) and the mutation of target OsACC-2 (+5G/C), the callus editing efficiency of the guided editor PE-P2 was 0, while the guided editor PE-P3 was able to achieve the mutation of both targets, 2.59% and 4.41% respectively; for the mutations (+1, +2, +5G/T) of target OsALS-1 and the mutations (+3, +5, +12A/G, G/C, T/C) of target OsACC-2, the callus editing efficiency of the guided editor PE-P2 was lower, 4.34% and 0.47% respectively, while the guided editor PE-P3 was able to increase the callus editing efficiency by 10.55% and 9.6% respectively; for the mutation (+1, +10, +14C/T, T/A, T/C) of target OsWaxy-1, the callus editing efficiency of the guided editor PE-P2 was 2.21%, while the callus editing efficiency of the guided editor PE-P3 was 17.16%. The guide editor PE-P3 can be seen to greatly improve the callus editing efficiency.
For target OsALS-1, the callus editing efficiencies of the RT-M template form (+1, +2, +5G/T) and the RT-S template form (+1G/T) of the lead editor PE-P2 were 4.34% and 0%, respectively, and the callus editing efficiencies of the RT-M template form (+1, +2, +5G/T) and the RT-S template form (+1G/T) of the lead editor PE-P3 were 10.55% and 2.59%, respectively; for target OsACC-2, the callus editing efficiencies of the RT-M template form (+3, +5, +12A/G, G/C, T/C) and the RT-S template form (+5G/C) of the guided editor PE-P2 were 0.47% and 0%, respectively, and the callus editing efficiencies of the RT-M template form (+3, +5, +12A/G, G/C, T/C) and the RT-S template form (+5G/C) of the guided editor PE-P3 were 9.6% and 4.41%, respectively; for target OsWaxy-1, the callus editing efficiencies of the RT-M template form (+1, +10, +14C/T, T/A, T/C) and the RT-S template form (+14T/C) of the guided editor PE-P2 were 2.21% and 0%, respectively; for target OsDEP1, the callus editing efficiencies of the RT-M template form (+8, +10, +12, +16A/C, C/A, T/G, T/A) and the RT-S template form (+8A/C) of the guide editor PE-P2 were 2.58% and 1.06%, respectively; for target OsALS-2, the callus editing efficiencies of the RT-M template form (+2, +5, +9C/A, G/A, C/T) and the RT-S template form (+9C/T) of the lead editor PE-P3 were 3.86% and 0%, respectively. The callus editing efficiency of the RT template design form of the RT-M is higher than that of the RT-S template form, so that the callus editing efficiency is greatly improved.
2. Taking the genome DNA of the rice positive T0 seedling obtained in the step 9 in the step one as a template for each vector, and carrying out PCR amplification on OsACC-2 targets by adopting a primer pair OsACC-2 to obtain a PCR amplification product; for OsACC-1 targets, carrying out PCR amplification by adopting a primer pair OsACC-1 to obtain PCR amplification products; for OsChalk targets, carrying out PCR amplification by adopting a primer pair OsChalk5 to obtain PCR amplification products; for OsDEP1 targets, carrying out PCR amplification by adopting a primer pair OsDEP to obtain PCR amplification products; for OsALS-2 targets, carrying out PCR amplification by adopting a primer pair OsALS-2 to obtain PCR amplification products; for OsWaxy-1 target, PCR amplification is carried out by adopting a primer pair OsWaxy-1 to obtain a PCR amplification product. All PCR amplified products were subjected to Sanger sequencing. The sequencing result is only analyzed for each pegRNA region, the number of T0 seedlings with base substitution of each target spot is counted, the editing efficiency of the guiding editor T0 seedlings is calculated, and the result is shown in Table 3.
The results show that for the mutation of target OsACC-2 (+5G/C) and the mutation of target OsDEP1 (+8A/C), the T0 shoot editing efficiency of the guide editor PE-P2 was 0% and 2.0%, respectively, while the guide editor PE-P3 was able to increase the mutation of both targets, the T0 shoot editing efficiency was 10.0% and 8.0%, respectively; for the mutations (+2, +5, +10T/A, G/C, A/G) of target OsACC, the mutations (+5, +14, +17G/C, T/C, A/T) of target OsChalk and the mutations (+1, +10, +14C/T, T/A, T/C) of target OsWaxy-1, the T0 shoot editing efficiency of the pilot editor PE-P2 was lower, 5.4%, 1.9% and 7.1%, respectively, while the pilot editor PE-P3 was able to increase the T0 shoot editing efficiency by 8.0%, 5.9% and 59.2%, respectively. The overall visual guide editor PE-P3 greatly improves the efficiency of editing the T0 seedlings.
For target OsACC-2, T0 shoot editing efficiencies of the RT-M template form (+3, +5, +12A/G, G/C, T/C) and the template form of RT-S (+5G/C) of the guide editor PE-P3 were 32.0% and 10.0%, respectively; for target OsACC-1, T0 seedlings were edited with 5.4% and 0% efficiency for the RT-M template form (+2, +5, +10T/A, G/C, A/G) and RT-S template form (+10A/G) of the boot editor PE-P2, respectively; t0 shoot editing efficiencies for the RT-M template form (+5, +14, +17G/C, T/C, A/T) and RT-S template form (+17A/T) of the target OsChalk guide editor PE-P2 were 1.9% and 0%, respectively; t0 shoot editing efficiencies for the RT-M template form (+8, +10, +12, +16A/C, C/A, T/G, T/A) and RT-S template form (+8A/C) of the target OsDEP 1-guided editor PE-P2 were 2.6% and 2.0%, respectively; t0 shoot editing efficiencies for the RT-M template form (+2, +5, +9C/A, G/A, C/T) and RT-S template form (+9C/T) of the target OsALS-2 guide editor PE-P3 were 8.0% and 0%, respectively; for target OsWaxy-1, the T0 shoot editing efficiencies of the RT-M template form (+1, +10, +14C/T, T/A, T/C) and the template form of RT-S (+14T/C) of the lead editor PE-P2 were 7.1% and 0%, respectively. The T0 seedling editing efficiency of the RT template design form of the RT-M is higher than that of the RT-S template form, and the T0 seedling editing efficiency is greatly improved.
TABLE 2
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Note that: mutant base count in RT template: the 1 st base from the 3' end of the RT template sequence is marked as +1.
TABLE 3 Table 3
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Note that: mutant base count in RT template: the 1 st base from the 3' end of the RT template sequence is marked as +1.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Sequence listing
<110> Academy of agriculture and forestry science in Beijing city
<120> Guide base editing system with improved gene editing efficiency and use thereof
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 17639
<212> DNA
<213> Artificial Sequence
<400> 1
ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg 60
cggacgtttt taatgtaggt accacctaaa tttccaagct tgtcgtgccc ctctctagag 120
ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt 180
gtttgaagtg cagtttatct atctttatac atatatttaa actttactct acgaataata 240
taatctatag tactacaata atatcagtgt tttagagaat catataaatg aacagttaga 300
catggtctaa aggacaattg agtattttga caacaggact ctacagtttt atctttttag 360
tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt 420
ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt 480
acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt 540
ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa 600
taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc 660
cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg 720
cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg 780
agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga 840
gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct 900
acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata 960
gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca 1020
accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct 1080
cccccccccc cctctctacc ttctctagat cggcgttccg gtccatggtt agggcccggt 1140
agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag 1200
cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac ttgccagtgt 1260
ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc gatttcatga 1320
ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc aatatatgcc 1380
gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg tgatgatgtg 1440
gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta cctggtggat 1500
ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga attgaagatg 1560
atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt actgatgcat 1620
atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc 1680
attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt attaattttg 1740
gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga tggaaatatc 1800
gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca tgatggcata 1860
tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata aacaagtatg 1920
ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca gctatatgtg 1980
gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt cttttgtcga 2040
tgctcaccct gttgtttggt gttacttctg cagtacgtaa gcatggacta caaggaccac 2100
gacggggatt acaaagacca cgacatagac tacaaggatg acgatgacaa aatggcaccg 2160
aagaaaaaaa ggaaggtcgg cggctccccg aagaaaaaaa ggaaggtcgg cggctccccg 2220
aagaaaaaaa ggaaggtcgg cggctccccg aagaaaaaaa ggaaggtcgg aatccatggc 2280
gttccagaat tcgacaagaa gtactccatc ggcctcgaca tcggcaccaa cagcgtcggc 2340
tgggcggtga tcaccgacga gtacaaggtc ccgtccaaga agttcaaggt cctgggcaac 2400
accgaccgcc actccatcaa gaagaacctc atcggcgccc tcctcttcga ctccggcgag 2460
acggcggagg cgacccgcct caagcgcacc gcccgccgcc gctacacccg ccgcaagaac 2520
cgcatctgct acctccagga gatcttctcc aacgagatgg cgaaggtcga cgactccttc 2580
ttccaccgcc tcgaggagtc cttcctcgtg gaggaggaca agaagcacga gcgccacccc 2640
atcttcggca acatcgtcga cgaggtcgcc taccacgaga agtaccccac tatctaccac 2700
cttcgtaaga agcttgttga ctctactgat aaggctgatc ttcgtctcat ctaccttgct 2760
ctcgctcaca tgatcaagtt ccgtggtcac ttccttatcg agggtgacct taaccctgat 2820
aactccgacg tggacaagct cttcatccag ctcgtccaga cctacaacca gctcttcgag 2880
gagaacccta tcaacgcttc cggtgtcgac gctaaggcga tcctttccgc taggctctcc 2940
aagtccaggc gtctcgagaa cctcatcgcc cagctccctg gtgagaagaa gaacggtctt 3000
ttcggtaacc tcatcgctct ctccctcggt ctgaccccta acttcaagtc caacttcgac 3060
ctcgctgagg acgctaagct tcagctctcc aaggatacct acgacgatga tctcgacaac 3120
ctcctcgctc agattggaga tcagtacgct gatctcttcc ttgctgctaa gaacctctcc 3180
gatgctatcc tcctttcgga tatccttagg gttaacactg agatcactaa ggctcctctt 3240
tctgcttcca tgatcaagcg ctacgacgag caccaccagg acctcaccct cctcaaggct 3300
cttgttcgtc agcagctccc cgagaagtac aaggagatct tcttcgacca gtccaagaac 3360
ggctacgccg gttacattga cggtggagct agccaggagg agttctacaa gttcatcaag 3420
ccaatccttg agaagatgga tggtactgag gagcttctcg ttaagcttaa ccgtgaggac 3480
ctccttagga agcagaggac tttcgataac ggctctatcc ctcaccagat ccaccttggt 3540
gagcttcacg ccatccttcg taggcaggag gacttctacc ctttcctcaa ggacaaccgt 3600
gagaagatcg agaagatcct tactttccgt attccttact acgttggtcc tcttgctcgt 3660
ggtaactccc gtttcgcttg gatgactagg aagtccgagg agactatcac cccttggaac 3720
ttcgaggagg ttgttgacaa gggtgcttcc gcccagtcct tcatcgagcg catgaccaac 3780
ttcgacaaga acctccccaa cgagaaggtc ctccccaagc actccctcct ctacgagtac 3840
ttcacggtct acaacgagct caccaaggtc aagtacgtca ccgagggtat gcgcaagcct 3900
gccttcctct ccggcgagca gaagaaggct atcgttgacc tcctcttcaa gaccaaccgc 3960
aaggtcaccg tcaagcagct caaggaggac tacttcaaga agatcgagtg cttcgactcc 4020
gtcgagatca gcggcgttga ggaccgtttc aacgcttctc tcggtaccta ccacgatctc 4080
ctcaagatca tcaaggacaa ggacttcctc gacaacgagg agaacgagga catcctcgag 4140
gacatcgtcc tcactcttac tctcttcgag gatagggaga tgatcgagga gaggctcaag 4200
acttacgctc atctcttcga tgacaaggtt atgaagcagc tcaagcgtcg ccgttacacc 4260
ggttggggta ggctctcccg caagctcatc aacggtatca gggataagca gagcggcaag 4320
actatcctcg acttcctcaa gtctgatggt ttcgctaaca ggaacttcat gcagctcatc 4380
cacgatgact ctcttacctt caaggaggat attcagaagg ctcaggtgtc cggtcagggc 4440
gactctctcc acgagcacat tgctaacctt gctggttccc ctgctatcaa gaagggcatc 4500
cttcagactg ttaaggttgt cgatgagctt gtcaaggtta tgggtcgtca caagcctgag 4560
aacatcgtca tcgagatggc tcgtgagaac cagactaccc agaagggtca gaagaactcg 4620
agggagcgca tgaagaggat tgaggagggt atcaaggagc ttggttctca gatccttaag 4680
gagcaccctg tcgagaacac ccagctccag aacgagaagc tctacctcta ctacctccag 4740
aacggtaggg atatgtacgt tgaccaggag ctcgacatca acaggctttc tgactacgac 4800
gtcgacgcca ttgttcctca gtctttcctt aaggatgact ccatcgacaa caaggtcctc 4860
acgaggtccg acaagaacag gggtaagtcg gacaacgtcc cttccgagga ggttgtcaag 4920
aagatgaaga actactggag gcagcttctc aacgctaagc tcattaccca gaggaagttc 4980
gacaacctca cgaaggctga gaggggtggc ctttccgagc ttgacaaggc tggtttcatc 5040
aagaggcagc ttgttgagac gaggcagatt accaagcacg ttgctcagat cctcgattct 5100
aggatgaaca ccaagtacga cgagaacgac aagctcatcc gcgaggtcaa ggtgatcacc 5160
ctcaagtcca agctcgtctc cgacttccgc aaggacttcc agttctacaa ggtccgcgag 5220
atcaacaact accaccacgc tcacgatgct taccttaacg ctgtcgttgg taccgctctt 5280
atcaagaagt accctaagct tgagtccgag ttcgtctacg gtgactacaa ggtctacgac 5340
gttcgtaaga tgatcgccaa gtccgagcag gagatcggca aggccaccgc caagtacttc 5400
ttctactcca acatcatgaa cttcttcaag accgagatca ccctcgccaa cggcgagatc 5460
cgcaagcgcc ctcttatcga gacgaacggt gagactggtg agatcgtttg ggacaagggt 5520
cgcgacttcg ctactgttcg caaggtcctt tctatgcctc aggttaacat cgtcaagaag 5580
accgaggtcc agaccggtgg cttctccaag gagtctatcc ttccaaagag aaactcggac 5640
aagctcatcg ctaggaagaa ggattgggac cctaagaagt acggtggttt cgactcccct 5700
actgtcgcct actccgtcct cgtggtcgcc aaggtggaga agggtaagtc gaagaagctc 5760
aagtccgtca aggagctcct cggcatcacc atcatggagc gctcctcctt cgagaagaac 5820
ccgatcgact tcctcgaggc caagggctac aaggaggtca agaaggacct catcatcaag 5880
ctccccaagt actctctttt cgagctcgag aacggtcgta agaggatgct ggcttccgct 5940
ggtgagctcc agaagggtaa cgagcttgct cttccttcca agtacgtgaa cttcctctac 6000
ctcgcctccc actacgagaa gctcaagggt tcccctgagg ataacgagca gaagcagctc 6060
ttcgtggagc agcacaagca ctacctcgac gagatcatcg agcagatctc cgagttctcc 6120
aagcgcgtca tcctcgctga cgctaacctc gacaaggtcc tctccgccta caacaagcac 6180
cgcgacaagc ccatccgcga gcaggccgag aacatcatcc acctcttcac gctcacgaac 6240
ctcggcgccc ctgctgcttt caagtacttc gacaccacca tcgacaggaa gcgttacacg 6300
tccaccaagg aggttctcga cgctactctc atccaccagt ccatcaccgg tctttacgag 6360
actcgtatcg acctttccca gcttggtggt gatagcggtg gctccagcgg tggtagcagc 6420
ggtagcgaaa ctccagggac ctcggaatcg gcgactccag aatccagtgg gggtagcagc 6480
ggcggatcca gcaccctcaa tatcgaggac gagtacaggc tgcatgagac atccaaggag 6540
ccggacgtgt cactcggctc tacatggctg agcgatttcc cacaggcctg ggcggagaca 6600
ggcggcatgg gcctcgcggt caggcaggcg ccgctcatca ttccactgaa ggcgacctcc 6660
acaccggtca gcatcaagca gtacccaatg tcacaggagg cacggctcgg catcaagcca 6720
cacattcaga ggctcctgga ccagggcatt ctggtccctt gccagagccc gtggaacacc 6780
cctctcctgc cggtgaagaa gcctggcaca aatgactaca ggccggtcca ggatctcagg 6840
gaggtgaaca agcgcgtcga ggatatccat ccgaccgtgc cgaacccata caatctcctg 6900
tcaggcctcc cgccatctca ccagtggtac accgtcctcg acctgaagga tgcgttcttc 6960
tgcctcaggc tgcatccaac aagccagcct ctcttcgcct tcgagtggcg cgatccagag 7020
atgggcattt caggccagct cacctggaca cggctgccac agggcttcaa gaactctcct 7080
accctcttca atgaggcgct ccatcgggac ctggccgatt tcaggatcca gcaccctgac 7140
ctcattctcc tgcagtacgt ggacgatctc ctgctcgccg cgacatcaga gctggattgc 7200
cagcagggca ccagggccct gctccagaca ctcggcaatc tgggctaccg ggcctctgcg 7260
aagaaggccc agatctgcca gaagcaggtg aagtacctcg gctacctgct caaggaggga 7320
cagaggtggc tgacagaggc aaggaaggag acagtcatgg gccagcctac cccgaagaca 7380
cctcggcagc tcagggagtt cctgggcaag gccggattct gcaggctctt cattccagga 7440
ttcgcggaga tggcggcgcc actctaccct ctgaccaagc cgggcacact gttcaactgg 7500
ggcccagacc agcagaaggc gtaccaggag attaagcagg cactgctcac agcacctgcg 7560
ctcggcctgc cggacctcac aaagccattc gagctgttcg tggatgagaa gcagggctac 7620
gcgaagggag tcctgacaca gaagctggga ccatggaggc gcccagtggc ctacctctca 7680
aagaagctcg acccagtggc ggccggatgg cctccgtgcc tgaggatggt ggcggccatt 7740
gccgtcctca ccaaggatgc cggcaagctg acaatgggcc agcctctcgt gattctggcg 7800
ccgcatgcgg tggaggccct ggtcaagcag ccacctgata ggtggctgtc caacgcgcgc 7860
atgacccact accaggccct gctcctggac acagataggg tccagttcgg accagtggtg 7920
gcactcaatc ctgccacact gctgccactc cctgaggagg gcctgcagca taactgcctc 7980
gatattctgg cggaggccca tggcacccgg ccagacctca cagatcagcc gctgccagac 8040
gccgatcaca cctggtacac agatggctca tctctcctgc aggagggcca gaggaaggcc 8100
ggagcagccg tgaccacaga gacagaggtc atctgggcaa aggccctccc agcgggcacc 8160
tcagcacaga gggccgagct cattgcactg acacaggcgc tcaagatggc cgagggcaag 8220
aagctgaatg tgtacacaga ctccaggtac gcattcgcca cagcacacat ccatggcgag 8280
atttacaggc ggaggggatg gctcacatca gagggaaagg agatcaagaa caaggatgag 8340
attctcgcgc tcctgaaggc cctcttcctg cctaagcgcc tgtcaatcat tcactgccca 8400
ggacatcaga agggacactc agccgaggca aggggaaata ggatggcaga ccaggcggcc 8460
aggaaggcag cgatcaccga gacaccagat acctccacac tcctgattga gaactccagc 8520
cctgacgatg acaaaatggc accgaagaaa aaaaggaagg tcggcggctc cccgaagaaa 8580
aaaaggaagg tcggcggctc cccgaagaaa aaaaggaagg tcggcggctc cccgaagaaa 8640
aaaaggaagg tcggaatcca tggcggatca ggagccacca acttctccct cctcaagcag 8700
gccggcgacg tggaggagaa cccgggccca atgaaaaagc ctgaactcac cgcgacgtct 8760
gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag 8820
ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta 8880
aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc 8940
gcgctcccga ttccggaagt gcttgacatt ggggagttta gcgagagcct gacctattgc 9000
atctcccgcc gttcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct 9060
gttctacaac cggtcgcgga ggctatggat gcgatcgctg cggccgatct tagccagacg 9120
agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc 9180
atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc 9240
agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa 9300
gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc 9360
ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc 9420
aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag 9480
cggaggcatc cggagcttgc aggatcgcca cgactccggg cgtatatgct ccgcattggt 9540
cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag 9600
ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc 9660
cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac 9720
cgacgcccca gcactcgtcc gagggcaaag aaatagacta gttcccgatc gttcaaacat 9780
ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 9840
atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 9900
gaggtgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 9960
aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagacct 10020
gcaggtggaa tcggcagcaa aggatttttt cctgtagttt tcccacaacc attttttacc 10080
atccgaatga taggatagga aaaatatcca agtgaacagt attcctataa aattcccgta 10140
aaaagcctgc aatccgaatg agccctgaag tctgaactag ccggtcacct gtacaggcta 10200
tcgagatgcc atacaagaga cggtagtagg aactaggaag acgatggttg attcgtcagg 10260
cgaaatcgtc gtcctgcagt cgcatctatg ggcctggacg gaatagggga aaaagttggc 10320
cggataggag ggaaaggccc aggtgcttac gtgcgaggta ggcctgggct ctcagcactt 10380
cgattcgttg gcaccggggt aggatgcaat agagagcaac gtttagtacc acctcgctta 10440
gctagagcaa actggactgc cttatatgcg cgggtgctgg cttggctgcc gatatctcgc 10500
tctcacattc cgtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt 10560
ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttttagga atctttaaac 10620
atacgaacag atcacttaaa gttcttctga agcaacttaa agttatcagg catgcatgga 10680
tcttggagga atcagatgtg cagtcaggga ccatagcaca agacaggcgt cttctactgg 10740
tgctaccagc aaatgctgga agccgggaac actgggtacg ttggaaacca cgtgtgatgt 10800
gaaggagtaa gataaactgt aggagaaaag catttcgtag tgggccatga agcctttcag 10860
gacatgtatt gcagtatggg ccggcccatt acgcaattgg acgacaacaa agactagtat 10920
tagtaccacc tcggctatcc acatagatca aagctggttt aaaagagttg tgcagatgat 10980
ccgtggcggg tatggtggtg caatggggtt tcagagctat gctggaaaca gcatagcaag 11040
ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcaaaccta 11100
tcctccaatt gcaccaccat ttttttttgg catgcaagct tggcactggc cgtcgtttta 11160
caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 11220
cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 11280
cgcagcctga atggcgaatg ctagagcagc ttgagcttgg atcagattgt cgtttcccgc 11340
cttcagttta aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag 11400
agcgtttatt agaataacgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat 11460
ttgtatgtgc atgccaacca cagggttccc ctcgggatca aagtactttg atccaacccc 11520
tccgctgcta tagtgcagtc ggcttctgac gttcagtgca gccgtcttct gaaaacgaca 11580
tgtcgcacaa gtcctaagtt acgcgacagg ctgccgccct gcccttttcc tggcgttttc 11640
ttgtcgcgtg ttttagtcgc ataaagtaga atacttgcga ctagaaccgg agacattacg 11700
ccatgaacaa gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc accgacgacc 11760
aggacttgac caaccaacgg gccgaactgc acgcggccgg ctgcaccaag ctgttttccg 11820
agaagatcac cggcaccagg cgcgaccgcc cggagctggc caggatgctt gaccacctac 11880
gccctggcga cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc 11940
tactggacat tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc ctggcagagc 12000
cgtgggccga caccaccacg ccggccggcc gcatggtgtt gaccgtgttc gccggcattg 12060
ccgagttcga gcgttcccta atcatcgacc gcacccggag cgggcgcgag gccgccaagg 12120
cccgaggcgt gaagtttggc ccccgcccta ccctcacccc ggcacagatc gcgcacgccc 12180
gcgagctgat cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc 12240
atcgctcgac cctgtaccgc gcacttgagc gcagcgagga agtgacgccc accgaggcca 12300
ggcggcgcgg tgccttccgt gaggacgcat tgaccgaggc cgacgccctg gcggccgccg 12360
agaatgaacg ccaagaggaa caagcatgaa accgcaccag gacggccagg acgaaccgtt 12420
tttcattacc gaagagatcg aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc 12480
cgcgcacgtc tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg atgccaagct 12540
ggcggcctgg ccggccagct tggccgctga agaaaccgag cgccgccgtc taaaaaggtg 12600
atgtgtattt gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt 12660
aaataaacaa atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc 12720
gggtcaggca agacgaccat cgcaacccat ctagcccgcg ccctgcaact cgccggggcc 12780
gatgttctgt tagtcgattc cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg 12840
gaagatcaac cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag 12900
gccatcggcc ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc ggacttggct 12960
gtgtccgcga tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag cccttacgac 13020
atatgggcca ccgccgacct ggtggagctg gttaagcagc gcattgaggt cacggatgga 13080
aggctacaag cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag 13140
gttgccgagg cgctggccgg gtacgagctg cccattcttg agtcccgtat cacgcagcgc 13200
gtgagctacc caggcactgc cgccgccggc acaaccgttc ttgaatcaga acccgagggc 13260
gacgctgccc gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact catttgagtt 13320
aatgaggtaa agagaaaatg agcaaaagca caaacacgct aagtgccggc cgtccgagcg 13380
cacgcagcag caaggctgca acgttggcca gcctggcaga cacgccagcc atgaagcggg 13440
tcaactttca gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag 13500
gcaagaccat taccgagctg ctatctgaat acatcgcgca gctaccagag taaatgagca 13560
aatgaataaa tgagtagatg aattttagcg gctaaaggag gcggcatgga aaatcaagaa 13620
caaccaggca ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc 13680
gtaagcggct gggttgtctg ccggccctgc aatggcactg gaacccccaa gcccgaggaa 13740
tcggcgtgac ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga 13800
cctggtggag aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc 13860
acgccccggt gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc 13920
gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt 13980
tttcgttccg atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc 14040
cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc 14100
agacgggcac gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga 14160
cctggtactg atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa 14220
gggagacaag cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg 14280
gcgagccgat ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac 14340
cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc 14400
cgagggtgaa gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga 14460
gtacatcgag atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc 14520
ggacgtgctg acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct 14580
ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat 14640
ctacgaacgc agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct 14700
gatcgggtca aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc 14760
gatcctagtc atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg 14820
tacggagcag atgctagggc aaattgccct agcaggggaa aaaggtcgaa aagttctctt 14880
tcctgtggat agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta 14940
cattgggaac ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa 15000
agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac 15060
ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc 15120
tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc 15180
tggccgctca aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc 15240
gccgtcgcca ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg 15300
atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 15360
cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 15420
gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc 15480
atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 15540
aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 15600
ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 15660
agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 15720
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 15780
caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 15840
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 15900
cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 15960
tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 16020
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 16080
cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 16140
tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 16200
tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 16260
caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 16320
aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 16380
cgaaaactca cgttaaggga ttttggtcat gcattctagg tactaaaaca attcatccag 16440
taaaatataa tattttattt tctcccaatc aggcttgatc cccagtaagt caaaaaatag 16500
ctcgacatac tgttcttccc cgatatcctc cctgatcgac cggacgcaga aggcaatgtc 16560
ataccacttg tccgccctgc cgcttctccc aagatcaata aagccactta ctttgccatc 16620
tttcacaaag atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg 16680
cttttccgtc tttaaaaaat catacagctc gcgcggatct ttaaatggag tgtcttcttc 16740
ccagttttcg caatccacat cggccagatc gttattcagt aagtaatcca attcggctaa 16800
gcggctgtct aagctattcg tatagggaca atccgatatg tcgatggagt gaaagagcct 16860
gatgcactcc gcatacagct cgataatctt ttcagggctt tgttcatctt catactcttc 16920
cgagcaaagg acgccatcgg cctcactcat gagcagattg ctccagccat catgccgttc 16980
aaagtgcagg acctttggaa caggcagctt tccttccagc catagcatca tgtccttttc 17040
ccgttccaca tcataggtgg tccctttata ccggctgtcc gtcattttta aatataggtt 17100
ttcattttct cccaccagct tatatacctt agcaggagac attccttccg tatcttttac 17160
gcagcggtat ttttcgatca gttttttcaa ttccggtgat attctcattt tagccattta 17220
ttatttcctt cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag 17280
acgaactcca attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt 17340
tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaaccg 17400
cggtgatcac aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag 17460
atcatccgtg tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca 17520
tgagcaaagt ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc 17580
tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggct 17639
<210> 2
<211> 1367
<212> PRT
<213> Artificial Sequence
<400> 2
Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 3
<211> 677
<212> PRT
<213> Artificial Sequence
<400> 3
Thr Leu Asn Ile Glu Asp Glu Tyr Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asn Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Phe Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Trp Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro
675
<210> 4
<211> 341
<212> PRT
<213> Artificial Sequence
<400> 4
Met Lys Lys Pro Glu Leu Thr Ala Thr Ser Val Glu Lys Phe Leu Ile
1 5 10 15
Glu Lys Phe Asp Ser Val Ser Asp Leu Met Gln Leu Ser Glu Gly Glu
20 25 30
Glu Ser Arg Ala Phe Ser Phe Asp Val Gly Gly Arg Gly Tyr Val Leu
35 40 45
Arg Val Asn Ser Cys Ala Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr
50 55 60
Arg His Phe Ala Ser Ala Ala Leu Pro Ile Pro Glu Val Leu Asp Ile
65 70 75 80
Gly Glu Phe Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg Arg Ser Gln
85 90 95
Gly Val Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu Pro Ala Val Leu
100 105 110
Gln Pro Val Ala Glu Ala Met Asp Ala Ile Ala Ala Ala Asp Leu Ser
115 120 125
Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro Gln Gly Ile Gly Gln Tyr
130 135 140
Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile Ala Asp Pro His Val Tyr
145 150 155 160
His Trp Gln Thr Val Met Asp Asp Thr Val Ser Ala Ser Val Ala Gln
165 170 175
Ala Leu Asp Glu Leu Met Leu Trp Ala Glu Asp Cys Pro Glu Val Arg
180 185 190
His Leu Val His Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn
195 200 205
Gly Arg Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp
210 215 220
Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu Ala
225 230 235 240
Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg His Pro Glu Leu
245 250 255
Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met Leu Arg Ile Gly Leu Asp
260 265 270
Gln Leu Tyr Gln Ser Leu Val Asp Gly Asn Phe Asp Asp Ala Ala Trp
275 280 285
Ala Gln Gly Arg Cys Asp Ala Ile Val Arg Ser Gly Ala Gly Thr Val
290 295 300
Gly Arg Thr Gln Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly
305 310 315 320
Cys Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr Arg
325 330 335
Pro Arg Ala Lys Lys
340
<210> 5
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 5
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 6
<211> 17636
<212> DNA
<213> Artificial Sequence
<400> 6
ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg 60
cggacgtttt taatgtaggt accacctaaa tttccaagct tgtcgtgccc ctctctagag 120
ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt 180
gtttgaagtg cagtttatct atctttatac atatatttaa actttactct acgaataata 240
taatctatag tactacaata atatcagtgt tttagagaat catataaatg aacagttaga 300
catggtctaa aggacaattg agtattttga caacaggact ctacagtttt atctttttag 360
tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt 420
ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt 480
acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt 540
ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa 600
taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc 660
cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg 720
cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg 780
agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga 840
gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct 900
acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata 960
gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca 1020
accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct 1080
cccccccccc cctctctacc ttctctagat cggcgttccg gtccatggtt agggcccggt 1140
agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag 1200
cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac ttgccagtgt 1260
ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc gatttcatga 1320
ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc aatatatgcc 1380
gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg tgatgatgtg 1440
gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta cctggtggat 1500
ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga attgaagatg 1560
atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt actgatgcat 1620
atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc 1680
attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt attaattttg 1740
gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga tggaaatatc 1800
gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca tgatggcata 1860
tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata aacaagtatg 1920
ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca gctatatgtg 1980
gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt cttttgtcga 2040
tgctcaccct gttgtttggt gttacttctg cagtacgtaa gcatggacta caaggaccac 2100
gacggggatt acaaagacca cgacatagac tacaaggatg acgatgacaa aatggcaccg 2160
aagaaaaaaa ggaaggtcgg cggctccccg aagaaaaaaa ggaaggtcgg cggctccccg 2220
aagaaaaaaa ggaaggtcgg cggctccccg aagaaaaaaa ggaaggtcgg aatccatggc 2280
gttccagaaa ccctcaatat cgaggacgag tacaggctgc atgagacatc caaggagccg 2340
gacgtgtcac tcggctctac atggctgagc gatttcccac aggcctgggc ggagacaggc 2400
ggcatgggcc tcgcggtcag gcaggcgccg ctcatcattc cactgaaggc gacctccaca 2460
ccggtcagca tcaagcagta cccaatgtca caggaggcac ggctcggcat caagccacac 2520
attcagaggc tcctggacca gggcattctg gtcccttgcc agagcccgtg gaacacccct 2580
ctcctgccgg tgaagaagcc tggcacaaat gactacaggc cggtccagga tctcagggag 2640
gtgaacaagc gcgtcgagga tatccatccg accgtgccga acccatacaa tctcctgtca 2700
ggcctcccgc catctcacca gtggtacacc gtcctcgacc tgaaggatgc gttcttctgc 2760
ctcaggctgc atccaacaag ccagcctctc ttcgccttcg agtggcgcga tccagagatg 2820
ggcatttcag gccagctcac ctggacacgg ctgccacagg gcttcaagaa ctctcctacc 2880
ctcttcaatg aggcgctcca tcgggacctg gccgatttca ggatccagca ccctgacctc 2940
attctcctgc agtacgtgga cgatctcctg ctcgccgcga catcagagct ggattgccag 3000
cagggcacca gggccctgct ccagacactc ggcaatctgg gctaccgggc ctctgcgaag 3060
aaggcccaga tctgccagaa gcaggtgaag tacctcggct acctgctcaa ggagggacag 3120
aggtggctga cagaggcaag gaaggagaca gtcatgggcc agcctacccc gaagacacct 3180
cggcagctca gggagttcct gggcaaggcc ggattctgca ggctcttcat tccaggattc 3240
gcggagatgg cggcgccact ctaccctctg accaagccgg gcacactgtt caactggggc 3300
ccagaccagc agaaggcgta ccaggagatt aagcaggcac tgctcacagc acctgcgctc 3360
ggcctgccgg acctcacaaa gccattcgag ctgttcgtgg atgagaagca gggctacgcg 3420
aagggagtcc tgacacagaa gctgggacca tggaggcgcc cagtggccta cctctcaaag 3480
aagctcgacc cagtggcggc cggatggcct ccgtgcctga ggatggtggc ggccattgcc 3540
gtcctcacca aggatgccgg caagctgaca atgggccagc ctctcgtgat tctggcgccg 3600
catgcggtgg aggccctggt caagcagcca cctgataggt ggctgtccaa cgcgcgcatg 3660
acccactacc aggccctgct cctggacaca gatagggtcc agttcggacc agtggtggca 3720
ctcaatcctg ccacactgct gccactccct gaggagggcc tgcagcataa ctgcctcgat 3780
attctggcgg aggcccatgg cacccggcca gacctcacag atcagccgct gccagacgcc 3840
gatcacacct ggtacacaga tggctcatct ctcctgcagg agggccagag gaaggccgga 3900
gcagccgtga ccacagagac agaggtcatc tgggcaaagg ccctcccagc gggcacctca 3960
gcacagaggg ccgagctcat tgcactgaca caggcgctca agatggccga gggcaagaag 4020
ctgaatgtgt acacagactc caggtacgca ttcgccacag cacacatcca tggcgagatt 4080
tacaggcgga ggggatggct cacatcagag ggaaaggaga tcaagaacaa ggatgagatt 4140
ctcgcgctcc tgaaggccct cttcctgcct aagcgcctgt caatcattca ctgcccagga 4200
catcagaagg gacactcagc cgaggcaagg ggaaatagga tggcagacca ggcggccagg 4260
aaggcagcga tcaccgagac accagatacc tccacactcc tgattgagaa ctccagccct 4320
agcggtggct ccagcggtgg tagcagcggt agcgaaactc cagggacctc ggaatcggcg 4380
actccagaat ccagtggggg tagcagcggc ggatccagcg acaagaagta ctccatcggc 4440
ctcgacatcg gcaccaacag cgtcggctgg gcggtgatca ccgacgagta caaggtcccg 4500
tccaagaagt tcaaggtcct gggcaacacc gaccgccact ccatcaagaa gaacctcatc 4560
ggcgccctcc tcttcgactc cggcgagacg gcggaggcga cccgcctcaa gcgcaccgcc 4620
cgccgccgct acacccgccg caagaaccgc atctgctacc tccaggagat cttctccaac 4680
gagatggcga aggtcgacga ctccttcttc caccgcctcg aggagtcctt cctcgtggag 4740
gaggacaaga agcacgagcg ccaccccatc ttcggcaaca tcgtcgacga ggtcgcctac 4800
cacgagaagt accccactat ctaccacctt cgtaagaagc ttgttgactc tactgataag 4860
gctgatcttc gtctcatcta ccttgctctc gctcacatga tcaagttccg tggtcacttc 4920
cttatcgagg gtgaccttaa ccctgataac tccgacgtgg acaagctctt catccagctc 4980
gtccagacct acaaccagct cttcgaggag aaccctatca acgcttccgg tgtcgacgct 5040
aaggcgatcc tttccgctag gctctccaag tccaggcgtc tcgagaacct catcgcccag 5100
ctccctggtg agaagaagaa cggtcttttc ggtaacctca tcgctctctc cctcggtctg 5160
acccctaact tcaagtccaa cttcgacctc gctgaggacg ctaagcttca gctctccaag 5220
gatacctacg acgatgatct cgacaacctc ctcgctcaga ttggagatca gtacgctgat 5280
ctcttccttg ctgctaagaa cctctccgat gctatcctcc tttcggatat ccttagggtt 5340
aacactgaga tcactaaggc tcctctttct gcttccatga tcaagcgcta cgacgagcac 5400
caccaggacc tcaccctcct caaggctctt gttcgtcagc agctccccga gaagtacaag 5460
gagatcttct tcgaccagtc caagaacggc tacgccggtt acattgacgg tggagctagc 5520
caggaggagt tctacaagtt catcaagcca atccttgaga agatggatgg tactgaggag 5580
cttctcgtta agcttaaccg tgaggacctc cttaggaagc agaggacttt cgataacggc 5640
tctatccctc accagatcca ccttggtgag cttcacgcca tccttcgtag gcaggaggac 5700
ttctaccctt tcctcaagga caaccgtgag aagatcgaga agatccttac tttccgtatt 5760
ccttactacg ttggtcctct tgctcgtggt aactcccgtt tcgcttggat gactaggaag 5820
tccgaggaga ctatcacccc ttggaacttc gaggaggttg ttgacaaggg tgcttccgcc 5880
cagtccttca tcgagcgcat gaccaacttc gacaagaacc tccccaacga gaaggtcctc 5940
cccaagcact ccctcctcta cgagtacttc acggtctaca acgagctcac caaggtcaag 6000
tacgtcaccg agggtatgcg caagcctgcc ttcctctccg gcgagcagaa gaaggctatc 6060
gttgacctcc tcttcaagac caaccgcaag gtcaccgtca agcagctcaa ggaggactac 6120
ttcaagaaga tcgagtgctt cgactccgtc gagatcagcg gcgttgagga ccgtttcaac 6180
gcttctctcg gtacctacca cgatctcctc aagatcatca aggacaagga cttcctcgac 6240
aacgaggaga acgaggacat cctcgaggac atcgtcctca ctcttactct cttcgaggat 6300
agggagatga tcgaggagag gctcaagact tacgctcatc tcttcgatga caaggttatg 6360
aagcagctca agcgtcgccg ttacaccggt tggggtaggc tctcccgcaa gctcatcaac 6420
ggtatcaggg ataagcagag cggcaagact atcctcgact tcctcaagtc tgatggtttc 6480
gctaacagga acttcatgca gctcatccac gatgactctc ttaccttcaa ggaggatatt 6540
cagaaggctc aggtgtccgg tcagggcgac tctctccacg agcacattgc taaccttgct 6600
ggttcccctg ctatcaagaa gggcatcctt cagactgtta aggttgtcga tgagcttgtc 6660
aaggttatgg gtcgtcacaa gcctgagaac atcgtcatcg agatggctcg tgagaaccag 6720
actacccaga agggtcagaa gaactcgagg gagcgcatga agaggattga ggagggtatc 6780
aaggagcttg gttctcagat ccttaaggag caccctgtcg agaacaccca gctccagaac 6840
gagaagctct acctctacta cctccagaac ggtagggata tgtacgttga ccaggagctc 6900
gacatcaaca ggctttctga ctacgacgtc gacgccattg ttcctcagtc tttccttaag 6960
gatgactcca tcgacaacaa ggtcctcacg aggtccgaca agaacagggg taagtcggac 7020
aacgtccctt ccgaggaggt tgtcaagaag atgaagaact actggaggca gcttctcaac 7080
gctaagctca ttacccagag gaagttcgac aacctcacga aggctgagag gggtggcctt 7140
tccgagcttg acaaggctgg tttcatcaag aggcagcttg ttgagacgag gcagattacc 7200
aagcacgttg ctcagatcct cgattctagg atgaacacca agtacgacga gaacgacaag 7260
ctcatccgcg aggtcaaggt gatcaccctc aagtccaagc tcgtctccga cttccgcaag 7320
gacttccagt tctacaaggt ccgcgagatc aacaactacc accacgctca cgatgcttac 7380
cttaacgctg tcgttggtac cgctcttatc aagaagtacc ctaagcttga gtccgagttc 7440
gtctacggtg actacaaggt ctacgacgtt cgtaagatga tcgccaagtc cgagcaggag 7500
atcggcaagg ccaccgccaa gtacttcttc tactccaaca tcatgaactt cttcaagacc 7560
gagatcaccc tcgccaacgg cgagatccgc aagcgccctc ttatcgagac gaacggtgag 7620
actggtgaga tcgtttggga caagggtcgc gacttcgcta ctgttcgcaa ggtcctttct 7680
atgcctcagg ttaacatcgt caagaagacc gaggtccaga ccggtggctt ctccaaggag 7740
tctatccttc caaagagaaa ctcggacaag ctcatcgcta ggaagaagga ttgggaccct 7800
aagaagtacg gtggtttcga ctcccctact gtcgcctact ccgtcctcgt ggtcgccaag 7860
gtggagaagg gtaagtcgaa gaagctcaag tccgtcaagg agctcctcgg catcaccatc 7920
atggagcgct cctccttcga gaagaacccg atcgacttcc tcgaggccaa gggctacaag 7980
gaggtcaaga aggacctcat catcaagctc cccaagtact ctcttttcga gctcgagaac 8040
ggtcgtaaga ggatgctggc ttccgctggt gagctccaga agggtaacga gcttgctctt 8100
ccttccaagt acgtgaactt cctctacctc gcctcccact acgagaagct caagggttcc 8160
cctgaggata acgagcagaa gcagctcttc gtggagcagc acaagcacta cctcgacgag 8220
atcatcgagc agatctccga gttctccaag cgcgtcatcc tcgctgacgc taacctcgac 8280
aaggtcctct ccgcctacaa caagcaccgc gacaagccca tccgcgagca ggccgagaac 8340
atcatccacc tcttcacgct cacgaacctc ggcgcccctg ctgctttcaa gtacttcgac 8400
accaccatcg acaggaagcg ttacacgtcc accaaggagg ttctcgacgc tactctcatc 8460
caccagtcca tcaccggtct ttacgagact cgtatcgacc tttcccagct tggtggtgat 8520
gacgatgaca aaatggcacc gaagaaaaaa aggaaggtcg gcggctcccc gaagaaaaaa 8580
aggaaggtcg gcggctcccc gaagaaaaaa aggaaggtcg gcggctcccc gaagaaaaaa 8640
aggaaggtcg gaatccatgg cggatcagga gccaccaact tctccctcct caagcaggcc 8700
ggcgacgtgg aggagaaccc gggcccaatg aaaaagcctg aactcaccgc gacgtctgtc 8760
gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 8820
gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 8880
agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 8940
ctcccgattc cggaagtgct tgacattggg gagtttagcg agagcctgac ctattgcatc 9000
tcccgccgtt cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 9060
ctacaaccgg tcgcggaggc tatggatgcg atcgctgcgg ccgatcttag ccagacgagc 9120
gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 9180
tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 9240
gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 9300
cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 9360
acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 9420
atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 9480
aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt 9540
gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 9600
cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 9660
agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 9720
cgccccagca ctcgtccgag ggcaaagaaa tagactagtt cccgatcgtt caaacatttg 9780
gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt 9840
tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag 9900
gtgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat 9960
atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagacctgca 10020
ggtggaatcg gcagcaaagg attttttcct gtagttttcc cacaaccatt ttttaccatc 10080
cgaatgatag gataggaaaa atatccaagt gaacagtatt cctataaaat tcccgtaaaa 10140
agcctgcaat ccgaatgagc cctgaagtct gaactagccg gtcacctgta caggctatcg 10200
agatgccata caagagacgg tagtaggaac taggaagacg atggttgatt cgtcaggcga 10260
aatcgtcgtc ctgcagtcgc atctatgggc ctggacggaa taggggaaaa agttggccgg 10320
ataggaggga aaggcccagg tgcttacgtg cgaggtaggc ctgggctctc agcacttcga 10380
ttcgttggca ccggggtagg atgcaataga gagcaacgtt tagtaccacc tcgcttagct 10440
agagcaaact ggactgcctt atatgcgcgg gtgctggctt ggctgccgat atctcgctct 10500
cacattccgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa ggctagtccg 10560
ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tttaggaatc tttaaacata 10620
cgaacagatc acttaaagtt cttctgaagc aacttaaagt tatcaggcat gcatggatct 10680
tggaggaatc agatgtgcag tcagggacca tagcacaaga caggcgtctt ctactggtgc 10740
taccagcaaa tgctggaagc cgggaacact gggtacgttg gaaaccacgt gtgatgtgaa 10800
ggagtaagat aaactgtagg agaaaagcat ttcgtagtgg gccatgaagc ctttcaggac 10860
atgtattgca gtatgggccg gcccattacg caattggacg acaacaaaga ctagtattag 10920
taccacctcg gctatccaca tagatcaaag ctggtttaaa agagttgtgc agatgatccg 10980
tggcgggtat ggtggtgcaa tggggtttca gagctatgct ggaaacagca tagcaagttg 11040
aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc aaacctatcc 11100
tccaattgca ccaccatttt tttttggcat gcaagcttgg cactggccgt cgttttacaa 11160
cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 11220
ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 11280
agcctgaatg gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt 11340
cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 11400
gtttattaga ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 11460
tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc 11520
gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt 11580
cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg 11640
tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca 11700
tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg 11760
acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga 11820
agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc 11880
ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac 11940
tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt 12000
gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg 12060
agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc 12120
gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg 12180
agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc 12240
gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc 12300
ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga 12360
atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt 12420
cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc 12480
gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc 12540
ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg 12600
tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa 12660
taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg 12720
tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat 12780
gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa 12840
gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc 12900
atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg 12960
tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata 13020
tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg 13080
ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt 13140
gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg 13200
agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac 13260
gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat 13320
gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac 13380
gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca 13440
actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca 13500
agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat 13560
gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa 13620
ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta 13680
agcggctggg ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg 13740
gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 13800
ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg 13860
ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc 13920
ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt 13980
cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt 14040
tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga 14100
cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct 14160
ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg 14220
agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg 14280
agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac 14340
gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga 14400
gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta 14460
catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga 14520
cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta 14580
ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta 14640
cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 14700
cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat 14760
cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac 14820
ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag ttctctttcc 14880
tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat 14940
tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga 15000
gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg 15060
cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac 15120
ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 15180
ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc 15240
gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 15300
acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 15360
atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg 15420
cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc 15480
agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 15540
gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 15600
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 15660
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 15720
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 15780
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 15840
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 15900
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 15960
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 16020
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 16080
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 16140
tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 16200
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 16260
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 16320
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 16380
aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa 16440
aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc 16500
gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata 16560
ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt 16620
cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt 16680
ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca 16740
gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg 16800
gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat 16860
gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga 16920
gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa 16980
gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg 17040
ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc 17100
attttctccc accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca 17160
gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta 17220
tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg 17280
aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca 17340
aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg 17400
tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 17460
atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 17520
gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 17580
ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggct 17636

Claims (11)

1. Use of the kit in any one of the following S1) -S4):
s1) editing plant or plant cell genome sequences;
s2) preparing an edited product of plant or plant cell genomic sequences;
S3) improving the editing efficiency of the plant or plant cell genome sequence;
s4) preparing a product for improving the editing efficiency of the plant or plant cell genome sequence;
the kit comprises a fusion protein or a biological material associated with the fusion protein, pegRNA or a biological material associated with the pegRNA;
The fusion protein is composed of reverse transcriptase, cas9 cutting enzyme, self-cutting oligopeptide and screening marker protein in sequence; the Cas9 nickase is Cas9nH840A; the Cas9nH840A is a protein with an amino acid sequence shown in a sequence 2; the reverse transcriptase is M-MLV RT; the M-MLV RT is a protein with an amino acid sequence shown in a sequence 3;
The pegRNA includes esgRNA, a reverse transcription template sequence, and a primer binding site sequence; the mutation bases introduced into the reverse transcription template sequence comprise a mutation base introduced into a target mutation site and additional mutation bases introduced into other sites except the target mutation site; the number of the mutation bases introduced into the target mutation site is one; the number of the additional mutation bases introduced at other sites than the target mutation site is two or three;
The plant is rice.
2. The use according to claim 1, characterized in that: the coding gene of the Cas9nH840A is a DNA molecule shown in 2293-6393 of the sequence 1.
3. The use according to claim 1, characterized in that: the coding gene of the M-MLV RT is a DNA molecule shown in 6493-8523 of a sequence 1.
4. The use according to claim 1, characterized in that: the screening marker protein is hygromycin phosphotransferase; the hygromycin phosphotransferase is a protein with an amino acid sequence shown in a sequence 4.
5. The use according to claim 4, characterized in that: the coding gene of the hygromycin phosphotransferase is a DNA molecule shown in 8731-9756 of a sequence 1.
6. The use according to claim 1, characterized in that: the self-cleaving oligopeptide is a 2A self-cleaving oligopeptide from a viral genome;
The 2A self-cleaving oligopeptide from the viral genome is a 2A self-cleaving oligopeptide from porcine teschovirus-1;
The amino acid sequence of the 2A self-cleaving oligopeptide from porcine teschovirus-1 is a protein shown in sequence 5.
7. The use according to claim 6, characterized in that: the coding gene of the 2A self-cleaving oligopeptide from the porcine teschovirus-1 is a DNA molecule shown in 8674-8730 of a sequence 1.
8. The use according to claim 1, characterized in that: the kit also includes esgRNA or biological material associated with the esgRNA.
9. The use according to claim 1, characterized in that: the editing of the genomic sequence is a base substitution of the genomic sequence.
T1) -T3) method as set forth in any one of:
T1) a method for editing a genomic sequence or a method for improving the efficiency of editing a genomic sequence of a plant or plant cell, comprising the steps of: allowing a plant or plant cell to express the fusion protein of claims 1-7 and pegRNA of claims 1-7;
T2) a method of editing a genomic sequence or a method of increasing the efficiency of editing a genomic sequence of a plant or plant cell, comprising the steps of: allowing a plant or plant cell to express the fusion protein of claims 1-7, pegRNA of claims 1-7, and esgRNA of claim 8;
T3) a method for preparing a plant mutant, comprising the steps of: editing the genome sequence of the plant or plant cell according to the method described in T1) or T2) to obtain a plant mutant;
The plant is rice.
11. The method according to claim 10, wherein: the editing of the genomic sequence is a base substitution of the genomic sequence.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951743A (en) * 2019-12-31 2020-04-03 北京市农林科学院 Method for improving plant gene replacement efficiency
CN111378051A (en) * 2020-03-25 2020-07-07 北京市农林科学院 PE-P2 guided editing system and application thereof in genome base editing
CN111748578A (en) * 2020-07-14 2020-10-09 北大荒垦丰种业股份有限公司 Plant guide template in-situ synthesis gene editing method and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3071440C (en) * 2017-07-31 2023-09-26 R. J. Reynolds Tobacco Company Methods and compositions for viral-based gene editing in plants
CN110157727A (en) * 2017-12-21 2019-08-23 中国科学院遗传与发育生物学研究所 Alkaloid edit methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951743A (en) * 2019-12-31 2020-04-03 北京市农林科学院 Method for improving plant gene replacement efficiency
CN111378051A (en) * 2020-03-25 2020-07-07 北京市农林科学院 PE-P2 guided editing system and application thereof in genome base editing
CN111748578A (en) * 2020-07-14 2020-10-09 北大荒垦丰种业股份有限公司 Plant guide template in-situ synthesis gene editing method and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A design optimized prime editor with expanded scope and capability in plants;Xu Wen等;nature plants;20211223;第8卷;第45-52页 *
Search-and-replace genome editing without double-strand breaks or donor DNA;Anzalone等;Nature;第576卷;第149–157页 *
秦瑞英 ; 魏鹏程 ; .Prime editing引导植物基因组精确编辑新局面.遗传.(06),第519-523页. *

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