CN110029096A - A kind of adenine base edit tool and application thereof - Google Patents

A kind of adenine base edit tool and application thereof Download PDF

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CN110029096A
CN110029096A CN201910382569.1A CN201910382569A CN110029096A CN 110029096 A CN110029096 A CN 110029096A CN 201910382569 A CN201910382569 A CN 201910382569A CN 110029096 A CN110029096 A CN 110029096A
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黄诗圣
李向阳
黄行许
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University of Shanghai for Science and Technology
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Abstract

The present invention relates to field of biotechnology, more particularly to a kind of adenine base edit tool and application thereof.The present invention provides a kind of fusion protein, including ecTadA-ecTadA* dimer fragment and SpCas9-NG D10A nickase segment, the ecTadA-ecTadA* dimer fragment include ecTad segment and ecTadA* segment.Fusion protein provided by the present invention can be with NG for PAM sequence, it holds 4-7 A to sport G sgRNA 5 ', the target site of base editor can be increased, furthermore, the fusion protein also has the advantages that editor's accuracy is high, low adjacent to missing the target, and has good industrialization prospect.

Description

A kind of adenine base edit tool and application thereof
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of adenine base edit tool and application thereof.
Background technique
CRISPR/Cas9 is the gene editing technology being most widely used at present.Cas9 arrival refers under the guidance of sgRNA Determine region and play digestion activity, is cut between the upstream PAM 3bp and 4bp.The double-strand of DNA is caused after CRISPR/Cas9 cutting Be broken (DSB), excite the DNA repair mechanism of itself, be broadly divided into HDR (Homologous Directly Repair, it is homologous heavy Group is repaired) and NHEJ (Non-Homologous End Join, non-homogeneous recombination end are repaired).HDR can be carried out quasi- using template Really repair, the result that NHEJ is repaired is randomly incorporated into insertion or missing, NHEJ in repair process in the highest flight.
The appearance of CRISPR/Cas9 is so that genetic manipulation is very convenient, but is randomly incorporated into insertion or missing simultaneously by NHEJ It can not achieve the accurate editor to genome, and the carrier or single stranded DNA (Single- of homologous recombination be provided after cutting Stranded Donor Oligonucleotide, ssODN) method, low efficiency and take considerable time.And Cas9 is cut Caused by DSB may cause the large fragment deletion of genome, leave security risk.
The David Liu et al. report of Harvard University is melted using the Cas9D10Anickase (nCas9) that RuvC structural domain inactivates The method for closing deaminase may be implemented to carry out point mutation (C-to-T or A-to-G) to genome single base, and not cause DSB, have Cytosine base edit tool (Cytosine Base Editor, CBE) and adenine base edit tool (Adenine Base Editor, ABE) two kinds.
The fusion protein of cytosine deaminase/adenine deaminase and nCas9 reach target site under the guidance of sgRNA And the DNA chain complementary with sgRNA is combined.Cytosine deaminase cytimidine C a certain range of to periphery, which carries out deamination, to be become Uracil U, U can be with adenine A complementary pairings, and by the duplication of DNA, U is eventually replaced the complementary pairing base T by A； Similar, adenine deaminase adenine A a certain range of to periphery, which carries out deamination, becomes hypoxanthine I, and I can be with cytimidine C complementary pairing, by the duplication of DNA, I is eventually replaced the complementary pairing bases G by C.To reach C-to-T or A-to- The purpose of G.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of adenine base edit tools And application thereof, for solving the problems of the prior art.
In order to achieve the above objects and other related objects, one aspect of the present invention provides a kind of fusion protein, including ecTadA- EcTadA* dimer fragment and SpCas9-NG D10A nickase segment, the ecTadA-ecTadA* dimer fragment packet Include ecTad segment and ecTadA* segment.
In some embodiments of the present invention, the amino acid sequence of the ecTadA segment includes:
A) amino acid sequence as shown in SEQ ID NO.57；Or,
B) with amino acid sequence of the SEQ ID NO.57 with 80% or more sequence similarity and with ammonia defined by a) The function of base acid sequence is preferably capable forming dimer with ecTadA* segment and dimer is living with adenine deaminase Property.
In some embodiments of the present invention, the amino acid sequence of the ecTadA* segment includes:
C) amino acid sequence as shown in SEQ ID NO.58；Or,
D) with amino acid sequence of the SEQ ID NO.58 with 80% or more sequence similarity and with ammonia defined by c) The function of base acid sequence is preferably capable forming dimer with ecTadA segment and dimer has adenine deaminase activity.
In some embodiments of the present invention, the amino acid sequence packet of the SpCas9-NG D10A nickase segment It includes:
E) amino acid sequence as shown in SEQ ID NO.59；Or,
F) with amino acid sequence of the SEQ ID NO.59 with 80% or more sequence similarity and with ammonia defined by e) The function of base acid sequence is preferably capable identification NG as PAM.
In some embodiments of the present invention, the fusion protein is from 5 ' ends to 3 ' ends successively including ecTadA-ecTadA* Dimer fragment and SpCas9-NG D10A nickase segment.
In some embodiments of the present invention, the ecTadA-ecTadA* dimer fragment is successively wrapped from 5 ' ends to 3 ' ends Include ecTad segment and ecTadA* segment.
In some embodiments of the present invention, the fusion protein further includes nuclear localization signal segment, it is preferred that the core Positioning signal segment be located at ecTadA-ecTadA* dimer fragment and SpCas9-NG D10A nickase segment 5 ' ends and/ Or 3 ' end, it is preferred that the amino acid sequence of the nuclear localization signal segment is as shown in SEQ ID NO.60.
In some embodiments of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID No.61.
Another aspect of the present invention provides a kind of isolated polynucleotides, encodes the fusion protein.
Another aspect of the present invention provides a kind of construct, and the construct contains the isolated polynucleotides.
Another aspect of the present invention provides a kind of expression system, and the expression system contains in the construct or genome It is integrated with the polynucleotides.
In some embodiments of the present invention, the host cell of the expression system is selected from eukaryocyte or prokaryotic cell, It is preferably selected from mouse cell, people's cell, it is thin to be more preferably selected from mouse brain nerve oncocyte, human embryonic kidney cell or human cervical carcinoma Born of the same parents are more preferably selected from N2a cell, HEK293FT cell or Hela cell.
Another aspect of the present invention provide the fusion protein, the isolated polynucleotides, the construct or Purposes of the expression system in gene editing.
In some embodiments of the present invention, the purposes is specially the purposes in Eukaryotic gene editing.
Another aspect of the present invention provides a kind of base editor system, including the fusion protein, the base editosome System further includes sgRNA.
Another aspect of the present invention provides a kind of gene editing method, comprising: passes through the fusion protein or the alkali Base editor's system carries out gene editing.
Detailed description of the invention
Fig. 1 is shown as the ABEmax-NG plasmid construct schematic diagram that the building of embodiment 1 obtains.
Fig. 2 is shown as 2 experimental result schematic diagram of the embodiment of the present invention, wherein a is eGFP reporting system Schematic diagram；B is microscope photo result of this report system on HEK293FT cell；C is flow cytometer detection result；D is Sanger sequencing result.
Fig. 3 is shown as 3 experimental result schematic diagram of the embodiment of the present invention, wherein a is for ABEmax-NG to 16 on N2a cell The deep-seq sequencing analysis result of a endogenous gene site editor；B is the base distribution ratio of corresponding editing sites；C is Corresponding mutation efficiency, mutation accessory substance and indel statistics；D is for ABEmax-NG to 4 endogenous genes on mice embryonic The deep-seq sequencing analysis result of site editor；E is corresponding mutation efficiency, mutation accessory substance and indel statistics.
Fig. 4 is shown as 3 experimental result schematic diagram of the embodiment of the present invention, wherein a is for ABEmax-NG to 16 on N2a cell The neighbouring result of missing the target of a endogenous gene site editor；B is that ABEmax-NG compiles 4 endogenous gene sites on mice embryonic The neighbouring result of missing the target collected.
Fig. 5 is shown as 4 experimental result schematic diagram of the embodiment of the present invention, wherein a be using ABEmax-NG on N2a cell To the edited result of the splice site of 4 endogenous genes；B is corresponding RNA montage testing result；C is corresponding Sanger survey Sequence is as a result, verify emerging montage hypotype.
Fig. 6 is shown as 5 experimental result schematic diagram of the embodiment of the present invention, wherein a is to obtain BBS2 base using ABEmax-NG Because of the schematic diagram of acceptor splicing site mutation mouse；B is the genotype identification result for being mutated mouse；C is the base for being mutated mouse different tissues Because of type qualification result；D is the RNA hypotype testing result for being mutated mouse different tissues.
Specific embodiment
Inventor passes through a large amount of pilot studys, provides a kind of fusion protein, and the fusion protein is a kind of New adenine base edit tool, the fusion protein can identify that NG as PAM, has widened the targeting model of base editor It encloses, completes the present invention on this basis.
First aspect present invention provides a kind of fusion protein, including ecTadA-ecTadA* dimer fragment and SpCas9- NGD10A nickase segment, the ecTadA-ecTadA* dimer fragment include ecTad segment and ecTadA* segment.Institute Stating fusion protein can match with the sgRNA of targeting target area, realize to sgRNA in target area with NG for PAM sequence The efficient base editor of the A-to-G at 5 ' 4-7, ends, and the accuracy being mutated is high, it is low adjacent to missing the target,
In fusion protein provided by the present invention, the amino acid sequence of the ecTadA segment may include: a) such as SEQ Amino acid sequence shown in ID NO.57；Or, b) having the amino acid sequence of 80% or more sequence similarity with SEQ ID NO.57 Column and have the function of a) defined by amino acid sequence.Specifically, it is described b) in amino acid sequence refer specifically to: such as SEQ Amino acid sequence shown in ID No.57 (specifically can be 1-50,1-30 by replacing, lacking or add one or more A, 1-20,1-10,1-5,1-3,1,2 or 3) obtained from amino acid, or in the end N- and/or C- End addition it is one or more (specifically can be 1-50,1-30,1-20,1-10,1-5,1-3,1,2, Or 3) obtained from amino acid, and have the function of the polypeptide piece of amino acid polypeptide fragment as shown in SEQ ID No.57 Section has that adenine deaminase is active for example, it may be having and can form dimer and dimer with ecTadA* segment, more It specifically can be the function that adenine (adenine, A) deamination is generated to hypoxanthine (hypoxanthine, I).It is described b) in Amino acid sequence can with SEQ ID No.57 have 80%, 85%, 90%, 93%, 95%, 97% or 99% or more phase Like property.
In fusion protein provided by the present invention, the amino acid sequence of the ecTadA* segment may include: c) such as SEQ Amino acid sequence shown in ID NO.58；Or, d) having the amino acid sequence of 80% or more sequence similarity with SEQ ID NO.58 Column and have the function of c) defined by amino acid sequence.Specifically, it is described d) in amino acid sequence refer specifically to: such as SEQ Amino acid sequence shown in ID No.58 (specifically can be 1-50,1-30 by replacing, lacking or add one or more A, 1-20,1-10,1-5,1-3,1,2 or 3) obtained from amino acid, or in the end N- and/or C- End addition it is one or more (specifically can be 1-50,1-30,1-20,1-10,1-5,1-3,1,2, Or 3) obtained from amino acid, and have the function of the polypeptide piece of amino acid polypeptide fragment as shown in SEQ ID No.58 Section has that adenine deaminase is active for example, it may be having and can form dimer and dimer with ecTadA segment, more It specifically can be the function that adenine (adenine, A) deamination is generated to hypoxanthine (hypoxanthine, I).It is described d) in Amino acid sequence can with SEQ ID No.58 have 80%, 85%, 90%, 93%, 95%, 97% or 99% or more phase Like property.
In fusion protein provided by the present invention, the amino acid sequence of the SpCas9-NG D10A nickase segment can To include: the e) amino acid sequence as shown in SEQ ID NO.59；Or, f) there is 80% or more sequence phase with SEQ ID NO.59 Like property amino acid sequence and have the function of e) defined by amino acid sequence.Specifically, it is described f) in amino acid sequence Refer specifically to: the amino acid sequence as shown in SEQ ID No.59 by replacing, missing or addition is one or more (specifically can be with It is 1-50,1-30,1-20,1-10,1-5,1-3,1,2 or 3) obtained from amino acid, or in N- One or more is added in end and/or the end C- (specifically can be 1-50,1-30 is a, 1-20 is a, 1-10 is a, 1-5 is a, 1-3 A, 1,2 or 3) obtained from amino acid, and the function with amino acid polypeptide fragment as shown in SEQ ID No.59 Can polypeptide fragment, for example, it may be function of the NG as PAM can be identified, specifically can be can using NG sequence as PAM, and can be matched with sgRNA the and ecTadA-ecTadA* dimer fragment in selectively targeted site, realize target spot SgRNA 5 ' holds the base editor of 4-7 A-to-G in region.It is described f) in amino acid sequence can be with SEQ ID No.59 With 80%, 85%, 90%, 93%, 95%, 97% or 99% or more similitude.The targeting of CRISPR/Cas9 system is known Not it is generally necessary to have space before sequence adjacent to motif (protospacer adjacent motif, PAM) beside target site, make It is frequently used for the Cas9 enzyme of genome editor the most for one kind, from streptococcus pyogenes (Streptococcus pyogenes) Cas9 (SpCas9) can only identify the PAM of NGG sequence, which limits the range that can be targeted in genome, and this hair SpCas9-NG D10A nickase segment in bright can identify NG sequence as PAM.
In fusion protein provided by the present invention, the substitution, missing or addition can be conserved amino acid substitution. " the conserved amino acid substitution " can specifically refer to that amino acid residue is replaced by other amino acid residues with similar side chain The case where.Amino acid residue families with similar side chain should be to those skilled in the art it is known, for example, can To be including but not limited to basic side chain (such as lysine, arginine, histidine), acid side-chain (such as aspartic acid, paddy ammonia Acid), uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, half Cystine), non-polar sidechain (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, first sulphur ammonia Acid, tryptophan) isoleucine) and the families such as aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).Conservative Amino acid substitution more specifically can include but is not limited in following table listed concrete condition, in table 1 (amino acid similarity matrix) Two amino acid of digital representation between similarity, be considered that conserved amino acid replaces when number is more than or equal to 0, table 2 is The scheme that illustrative conserved amino acid replaces.
Table 1
C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6
K -5 -2 -1 0 -1 0 0 0 1 1 0 5
H -3 -2 0 -1 -1 -1 1 1 2 3 6
Q -5 -1 0 -1 0 -1 2 2 1 4
N -4 0 -1 1 0 0 2 1 2
E -5 0 -1 0 0 0 3 4
D -5 1 -1 0 0 0 4
T -2 0 0 1 1 3
A -2 1 1 1 2
S 0 1 1 1
P -3 -1 6
G -3 5
C 12
Table 2
It can also include nuclear localization signal segment (NLS), the nuclear localization signal in fusion protein provided by the present invention Segment can be located at the N-terminal of ecTadA-ecTadA* dimer fragment, can also be located at SpCas9-NG D10A nickase piece The C-terminal of section.The nuclear localization signal segment may include the amino acid sequence as shown in SEQ ID NO.60.
In fusion protein provided by the present invention, it can successively include ecTadA- that the fusion protein, which is held from 5 ' ends to 3 ', EcTadA* dimer fragment and SpCas9-NG D10A nickase segment, the ecTadA-ecTadA* dimer fragment is certainly 5 ' ends to 3 ' ends can successively include ecTadA segment and ecTadA* segment.In a specific embodiment of the invention, the fusion The amino acid sequence of albumen is as shown in SEQ ID No.61.
Second aspect of the present invention provides a kind of isolated polynucleotides, merges egg provided by coding first aspect present invention It is white.
Third aspect present invention provides a kind of construct, and the construct contains separation provided by second aspect of the present invention Polynucleotides.The construct can usually be constructed by the way that the isolated polynucleotides are inserted into suitable expression vector It obtains, suitable expression vector may be selected in those skilled in the art, for example, the expression vector can be including but not limited to PCMV expression vector, pSV2 expression vector, pGL3 expression vector etc..
Fourth aspect present invention provides a kind of expression system, and the expression system contains provided by third aspect present invention The polynucleotides separated provided by the second aspect of the present invention of external source are integrated in construct or genome.The expression system It can be host cell, the host cell can express fusion protein as described above, and the fusion protein can be with sgRNA It matches, so as to which the fusion protein is navigated to target area, realizes the base editor of target area.Of the invention another In one specific embodiment, the host cell can be eukaryocyte and/or prokaryotic cell, more specifically can be mouse cell, People's cell etc. more specifically can be mouse brain nerve oncocyte, human embryonic kidney cell, human cervical carcinoma cell etc., more specifically can be with It is N2a cell, HEK293FT cell, Hela cell etc..
Fifth aspect present invention provides fusion protein provided by first aspect present invention or second aspect of the present invention is mentioned Table provided by construct provided by the isolated polynucleotides or third aspect present invention of confession or fourth aspect present invention Up to purposes of the system in gene editing, purposes in preferably Eukaryotic gene editing, the eucaryote specifically may be used To be metazoa, including but not limited to mouse etc. specifically can be.The purposes specifically can be including but not limited to by A to G base editor (more specifically in target area sgRNA 5 ' hold 4-7 A-to-G base editor), edit montage by Body/donor site is come building or the treatment of human diseases etc. for adjusting RNA montage, this tool being utilized to carry out mouse disease model. In a specific embodiment of the invention, gene to be edited can be CHRNE (ID:11448), SIX6 (ID:20476), ITPR1 (ID:16438)、TMEM67(ID:329795)、LMBR1(ID:56873)、NFIX(ID:18032)、DES(ID:13346)、 BHLHA9(ID:320522)、NDUFS1(ID:227197)、HOXD13(ID:15433)、AKR1C19(ID:432720)、LMNA (ID:16905)、WNT5A(ID:22418),、SUFU(ID:24069)、GJA8(ID:14616)、EYA1(ID:14048)、BBS2 (ID:67378), OFD1 (ID:237222), MYO7A (ID:17921), SEPN1 (ID:74777) etc..Another specific reality of the present invention It applies in example, object to be edited can be embryo, cell etc..
Sixth aspect present invention provides a kind of base editor system, including egg is merged provided by first aspect present invention White, the base editor system further includes sgRNA.Those skilled in the art can select according to the target editing area of gene The sgRNA in suitable targeting specific site.For example, the sequence of the sgRNA usually can be at least partly mutual with target area It mends, so as to be matched with the fusion protein, the fusion protein is navigated into target area, is realized in target area SgRNA 5 ' holds the base editor of 4-7 A-to-G, specifically can be adenine desamination reaction, i.e., compiles adenine (A) Collect is hypoxanthine (I).Base editor system provided by the present invention has greatly widened the range that genome can target, can be with Using NG sequence as PAM, the base of the A-to-G at 5 ' 4-7, ends in sgRNA target area is realized, and be mutated with very high essence Parasexuality is low adjacent to missing the target.In an of the invention specific embodiment, the sgRNA can target CHRNE, SIX6, ITPR1, TMEM67、LMBR1、NFIX、DES、BHLHA9、NDUFS1、HOXD13、AKR1C19、LMNA、WNT5A、SUFU、GJA8、EYA1、 The genes such as BBS2, OFD1, MYO7A, SEPN1.In another specific embodiment of the present invention, object to be edited can be embryo, thin Born of the same parents etc..
Seventh aspect present invention provides a kind of base edit methods, comprising: melts provided by first aspect through the invention Base editor system provided by hop protein or sixth aspect present invention carries out gene editing.For example, the gene editing method It may include: to cultivate expression system provided by fourth aspect present invention under proper condition, to express the fusion protein, The fusion protein can it is engaged therewith targeting target area sgRNA existing under the conditions of, to targeting regions carry out alkali Base editor.The method for providing condition existing for the sgRNA should be known to those skilled in the art, for example, Can be culture under proper condition can express the expression system of the sgRNA, and the expression system can be including containing It encodes and is integrated with the more of the coding sgRNA in the host cell or chromosome of the expression vector of the polynucleotides of the sgRNA The host cell of nucleotide.In a specific embodiment of the invention, the gene editing is outer-gene editor.
The present invention provides a kind of new adenine base by combining the SpCas9-NG for identifying NG PAM with ABE SgRNA 5 ' can be held 4-7 A to sport G by edit tool, fusion protein provided by the present invention with NG for PAM sequence, The target site that base editor can be increased possesses more for editing acceptor splicing site/donor site sgRNA, can be used for adjusting The gene number of section RNA montage more possesses bigger base editor's range in genome.In addition, the fusion protein also has Editor's accuracy height, neighbouring low advantage of missing the target, have good industrialization prospect.
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
ABEmax-NG plasmid is constructed first, passes through Mut Express II Fast Mutagenesis Kit V2 (Vazyme, C214-02) is by 7 amino acid mutation (R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/ T1337R ABEmax plasmid) is introduced, ABEmax plasmid is purchased from Addgene (#112095).Construct obtained ABEmax-NG plasmid Sequence is shown in annex sequence table SEQ ID.1.
Embodiment 2
In the present embodiment, the volume of ABEmax-NG is verified on HEK293FT using eGFP reporting system The ability of collecting.
The last 1.1 green fluorescent protein reporting system plasmid construction
It is green to enhancing by Mut Express II Fast Mutagenesis Kit V2 (Vazyme, C214-02) Fluorescence protein expression carrier introduce two base mutations, one be located at -63 codon of threonine third position, sport T, A or G, the mutation will not change the amino acid sequence of eGFP, but provide a variety of PAM sequences to test ABEmax- The identification of NG；Another is located at first of -69 codon of glutamine, sports T, converts thereof into a termination codon Son, so that eGFP be made to lose fluorescence.Construct obtained eGFP reporting system plasmid Sequence is shown in annex sequence table SEQ ID.2.
The building of 1.2sgRNA plasmid
Design sgRNA simultaneously synthesizes oligos, upstream sequence are as follows: 5 '-accgAGCACTACACGCCGTAGGTC-3 ' (SEQ ID NO.3), downstream sequence are as follows: 5 '-aaacGACCTACGGCGTGTAGTGCT-3 ' (SEQ ID NO.4), upstream and downstream sequence are logical Cross program (95 DEG C, 5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；4 DEG C of hold at) annealing, connection Onto pGL3-U6-sgRNA (Addgene#51133) carrier by BsaI (NEB:R0539L) linearisation.Linearisation system is such as Shown in lower: 2 μ g of pGL3-U6-sgRNA；6 μ L of buffer (NEB:R0539L)；BsaI 2μL；DdH2O polishing is to 60 μ L.37℃ Digestion is stayed overnight.Linked system is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, linearized vector 20ng, the oligo of annealing Segment (10 μM) 5 μ L, T4 ligase (NEB:M0202L) 0.5 μ L, ddH2O polishing to 10 μ, L.16 stay overnight by a DEG C connection.The load of connection Body chooses bacterium by conversion, identifies.Bacterium extraction plasmid (Axygene:AP-MN-P-250G) is shaken to positive colony and measures concentration.
The culture transfection and identification of 1.3 cells
HEK293FT cell (being purchased from ATCC) inoculated and cultured is in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein Streptomycin containing 1%Penicillin (v/v) (Gibco).Work as cell concentration When being 80%, liquid is changed with the DMEM culture medium of 10% serum, culture makes cell state restore best for 2 hours.The matter of every hole transfection The amount of grain is 1 μ g, sgRNA plasmid of ABEmax-NG plasmid, 0.5 μ g, the eGFP expression plasmid after mutation respectively 0.5μg.Plasmid is mixed in Opti-MEM (Gibco, 11058021) culture medium of 50 μ l.By the Lipofectamine of 2 μ l 2000 transfection reagents (Thermo, 11668019) are mixed into the Opti-MEM culture medium of 50 μ l and mix, and stand 5 minutes.It will be mixed with The Opti-MEM for being mixed with Lipofectamine 2000 is added in the Opti-MEM of plasmid, and piping and druming mixes at a slow speed, stands 20 minutes.It will The Opti-MEM for being mixed with plasmid and Lipofectamine 2000 is separately added into 12 orifice plates.With 10%FBS's after transfection 6 hours DMEM changes liquid.Transfection observes fluorescence under the microscope and takes pictures after 48 hours, and with Flow cytometry Fluorescence Ratio.Sorting GFP positive cell identifies genotype by cracking, and the ingredient of lysate is 50mM KCl, 1.5mM MgCl2,10mM Tris PH 8.0,0.5%Nonidet P-40,0.5%Tween 20,100g/ml protease K.Sequence near target spot is carried out PCR amplification identifies amplified production with Sanger sequencing after purification.Amplification system is as follows: 2Xbuffer (Vazyme, P505)25μL；dNTP 1μL；F(10pmol/μL)1μL；R(10pmol/μL)1μL；1 μ L of template；Archaeal dna polymerase (Vazyme, P505)0.5μL；DdH2O polishing is to 50 μ L.It amplifies the PCR product come to purify by following step: three times volume is added PCR-A (Axygen:AP-PCR-250G) crosses column, and centrifugation, 12000 revs/min are centrifuged 1 minute；700 μ L W2 are added, are centrifuged 1 point Clock；Waste liquid is abandoned, 700 μ LW2 are added, is centrifuged 1 minute；Waste liquid is abandoned, is dallied 1 minute；20 μ L water elutions are added.Correlated results such as Fig. 2 It is shown.ABEmax-NG can identify NG PAM in HEK293FT cell, and the enhancing fluorescin after repairing mutation keeps its extensive Multiple fluorescence.
Embodiment 3
In the present embodiment, endogenous gene site is compiled on N2a cell and mice embryonic using ABEmax-NG Volume.
The building of 2.1sgRNA plasmid
Select 16 endogenous mouse genes: CHRNE (ID:11448), SIX6 (ID:20476), ITPR1 (ID:16438), TMEM67(ID:329795)、LMBR1(ID:56873)、NFIX(ID:18032)、DES(ID:13346)、BHLHA9(ID: 320522)、NDUFS1(ID:227197)、HOXD13(ID:15433)、AKR1C19(ID:432720)、LMNA(ID:16905)、 WNT5A (ID:22418), SUFU (ID:24069), GJA8 (ID:14616), EYA1 (ID:14048) design sgRNA, used Oligos see annex sequence table SEQ ID.5-36.The building for carrying out sgRNA plasmid by 1.2.
Select 4 endogenous mouse genes: BBS2 (ID:67378), OFD1 (ID:237222), MYO7A (ID:17921), SEPN1 (ID:74777) designs sgRNA and synthesizes oligos, the PUC57- of linearisation is annealed and be connected to by 1.2 method On T7-sgRNA carrier (addgene:51132).Linearisation system is as described in 1.2.Used oligos is shown in annex sequence table SEQ ID.37-44。
The culture transfection and identification of 2.2 cells
N2a cell (being purchased from ATCC) is cultivated and is transfected by 1.3, and the plasmid amount of transfection is 1 μ g of ABEmax-NG, 0.5 μ g of sgRNA expression vector plasmid, using ABEmax as control.GFP positive cell is sorted after transfection 72 hours, is carried out by 1.3 Cracking, PCR amplification and purifying, product are sequenced with two generations and are identified.Correlated results is as shown in Figure 3 and Figure 4.ABEmax-NG can To identify NG PAM in N2a cell, endogenous gene site is efficiently edited, and accuracy is high, accessory substance is low, neighbouring misses the target It is low.
The in-vitro transcription of 2.3sgRNA
Using the PUC57-T7sgRNA of building as template, the segment containing sgRNA, the primer are as follows: F:5 '-are expanded TCTCGCGCGTTTCGGTGATGACGG-3'(SEQ ID.45)；R:5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTC- 3'(SEQ ID.46).Amplification system is as follows: 25 μ L of 2Xbuffer (Vazyme, P505)；dNTP 1μL；F(10pmol/μL)2μ L；R(10pmol/μL)2μL；Template 1ng；0.5 μ L of archaeal dna polymerase (Vazyme, P505)；DdH2O polishing is to 50 μ L.It amplifies The PCR product come is purified by following step: every 100 μ L volume adds 4 μ L RNAsecure (Life:AM7005)；60 DEG C 15 points Clock；The PCR-A (Axygen:AP-PCR-250G) that three times volume is added crosses column, and centrifugation, 12000 revs/min are centrifuged 1 minute；Add Enter 500 μ L W2, is centrifuged 1 minute；Idle running 1 minute；20 μ L are added without RNAase water elution.
It is transcribed using in-vitro transcription kit (Ambion, Life Technologies, AM1354), steps are as follows:
Reaction system are as follows: 1 μ L of reaction buffer；enzyme mix 1μL；A1μL；T 1μL；G 1μL；C 1μL； Template 800ng；H2O polishing is to 10 μ L.37 DEG C of 5 hours of reaction after above-mentioned system mixes.1 μ L DNase, 37 DEG C of reactions are added 15 minutes.Using the sgRNA of QIAquick Gel Extraction Kit (Ambion, Life Technologies, AM1908) recycling transcription, step is such as Under: it above walks reaction volume and 90 μ LElution solution transplanting 1.5ml EP pipe is added；350 μ L Binding are added Solution is mixed；The mixing of 250 μ L dehydrated alcohols is added；Upper prop；10000 revs/min are centrifuged 30 seconds, outwell waste liquid；It is added 500 μ L Washing solution, 10000 revs/min are centrifuged 30 seconds, outwell waste liquid；Idle running 1 minute；Collecting pipe is changed, is added 100 μ L Elution solution elution；It is mixed that 10 μ L ammonium acetates (Ambion, Life Technologies, AM1908) is added It is even；The mixing of 275 μ L dehydrated alcohols is added；- 20 DEG C are placed 30 minutes, while being prepared 70% ethyl alcohol and being placed -20 DEG C；Under 4 DEG C of environment 13000 revs/min are centrifuged 15 minutes.Supernatant is abandoned, 500 μ L, 70% ethyl alcohol is added；Centrifugation 5 minutes, siphons away waste liquid, dries 5 points Clock；The water dissolution of 20 μ L is added；1 μ L is taken to survey concentration.
The in-vitro transcription of 2.4ABEmax-NG and ABEmax
Plasmid enzyme restriction is recycled.This step is to linearize plasmid.System is as follows: 10 μ g of plasmid；buffer I (NEB:R0539L) 10 μ L；4 μ L (NEB:R0539L) of BbsI；H2O polishing is to 100 μ L.After mixing, 37 DEG C of digestions are stayed overnight.
The recycling of linearization plasmid.4 μ L RNAsecure (Life:AM7005), 60 DEG C of reactions 10 are added in digestion products Minute；It carries out operating remaining step using QIAquick Gel Extraction Kit (QIAGEN:28004), 5 times of volume buffer PB is added, cross column； 750 μ L buffer PE centrifugation is added；Idle running 1 minute；With 10 μ L water elutions, concentration is measured.
It is transcribed in vitro.Sequentially add system according to the requirement of kit (Invitrogen:AM1345): 1 μ g linearisation carries Body；10μL2XNTP/ARCA；Polishing is to 20 μ L water；2μL T7ezyme mix；2μL 10xreaction buffer.Mix it It reacts 2 hours for 37 DEG C afterwards.1 μ L DNasea is added to react 15 minutes.
Tailing.Transcription product carries out the stability that tailing processing guarantees transcript mRNA.Specific system is as follows: 20 μ L reaction produces Object；36μL H2O；20μL 5xE-PAP buffer；10μL 25mM MnCl2；10μL ATP solution；4μL PEP.Instead It is reacted 30 minutes for 37 DEG C after answering system to mix.
Recycling.(QIAGEN:74104) is carried out using QIAquick Gel Extraction Kit.Steps are as follows: above walking reaction product and 350 μ L are added buffer RLT；250 μ L dehydrated alcohols are added, cross column, centrifugation；500 μ L RPE are added, are centrifuged, 500 μ L RPE, centrifugation is added； Idle running；30 μ L water elutions are added.- 80 DEG C of preservations after measurement concentration.
The injection of 2.5 mice embryonics, in vitro culture and identification
To 1-cell embryo inject ABEmax-NG/ABEmax and sgRNA mixture, concentration be respectively 100ng/ μ L and 50ng/μL.In vitro culture arrive E4.5 days, condition of culture be KSOM culture solution (Millipore, MR-106-D), 37 DEG C, 5% CO2.Embryo transfer is managed to 200 μ L, adds 5 μ L alkaline lysis solution (200mM KOH/50mM dithiothreitol).65 DEG C be incubated for after ten minutes, add neutralization solution (900mM Tris-HCl, PH 8.3/300mM KCl/200mM HCl), 5 400 μM of μ L random primers (Genscript, Nanjing, China), 6 μ L 10x PCR buffer (Takara, Dalian, China), 3 μ L dNTPs (2.5mM) and 1 μ L Taq polymerase (Takara, Dalian, China), moisturizing to 60 μ L.PCR 50 circulation, it is each circulation include 92 DEG C 1 minute；Prolong within 2 minutes It stretches, temperature is with 10sec/degree from 37 DEG C to 55 DEG C；55 DEG C 4 minutes.Amplified production carries out purpose as pcr template, by 1.3 The PCR amplification of segment, purifying, purified product are sequenced with two generations and are identified, correlated results is as shown in Figure 3 and Figure 4.ABEmax- NG can identify NG PAM in mice embryonic, efficiently edit endogenous gene site, and accuracy is high, accessory substance is low, adjacent It closely misses the target low.
Embodiment 4
In the present embodiment, the RNA montage of endogenous gene is adjusted in N2a cell using ABEmax-NG.
The building of 3.1sgRNA plasmid
Select 4 endogenous mouse genes: BBS2 (ID:67378), OFD1 (ID:237222), MYO7A (ID:17921), SEPN1 (ID:74777), designs sgRNA, and used oligos is shown in annex sequence table SEQ ID.47-54.It is carried out by 1.2 The building of sgRNA plasmid.
The culture transfection and identification of 3.2 cells
N2a cell (being purchased from ATCC) is cultivated and is transfected by 1.3, and the plasmid amount of transfection is 1 μ g of ABEmax-NG, 0.5 μ g of sgRNA expression vector plasmid, using ABEmax as control.GFP positive cell is sorted after transfection 72 hours, as 24 holes It is cultivated in plate to covering with.Part cell is taken to be cracked by 1.3, PCR amplification and purifying, product are sequenced with two generations and are identified. Remaining cell mentions total serum IgE with TRIzol method, carries out reverse transcription with HiScript II Q RT SuperMix (Vazyme, R222), The PCR amplification for carrying out reverse transcription product by 1.3.The separation of RNA hypotype is carried out using agarose gel electrophoresis, isolated RNA is sub- Type is sequenced with Sanger and is identified.
Correlated results is as shown in Figure 5.ABEmax-NG can identify NG PAM in N2a cell, efficiently edit endogenous base Because of site splice site, to change the RNA montage of endogenous gene.
Embodiment 5
In the present embodiment, BBS2 splice site mutant mice is obtained using ABEmax-NG, it was demonstrated that ABEmax-NG can be at For the effective tool for making RNA splice model mouse.
The building of 4.1sgRNA plasmid
The sgRNA of design editing mouse BBS2 gene (ID:67378) splice site, used oligos are shown in annex sequence List SEQ ID.55-56.The building for carrying out PUC57-T7-sgRNA vector plasmid by 2.1.
4.2 being transcribed in vitro
The in-vitro transcription for carrying out sgRNA and ABEmax-NG by 2.3 and 2.4.
The injection of 4.3 mice embryonics and transplanting
The injection for carrying out mice embryonic by 2.5.It will be in the embryo transfer after injection to replace-conceive female rat (background: ICR strain).
The detection of the identification of 4.4 murine genes types and RNA montage
Mousetail is taken, extracts genomic DNA with phenol chloroform method, carries out PCR amplification and purifying by 1.3, product is used Sanger sequencing is identified.Take different tissues of mice, with phenol chloroform method extract genomic DNA, by 1.3 carry out PCR amplifications and Purifying, product are sequenced with two generations and are identified；Extraction, reverse transcription, amplification and the purifying for carrying out RNA by 3.2, product are surveyed with two generations Sequence is identified.Correlated results is as shown in Figure 6.BBS2 splice site mutation mouse is successfully obtained using ABEmax-NG, each The mutation and corresponding RNA montage of splice site can be detected in tissue.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.
Sequence table
<110>Shanghai Science and Technology Univ.
<120>a kind of adenine base edit tool and application thereof
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8811
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420
gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga agtcgagttt 480
agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga 540
gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac 600
aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga 660
ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca 720
tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca 780
cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg 840
aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc 900
gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc 960
gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc 1020
gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac 1080
gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg 1140
cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc 1200
atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg 1260
gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg 1320
atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac 1380
gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac 1440
cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc 1500
tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc 1560
ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 1620
cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 1680
atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1740
aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1800
ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1860
agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1920
gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1980
aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 2040
aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 2100
ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 2160
gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 2220
acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 2280
atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 2340
ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 2400
aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 2460
tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 2520
ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2580
gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2640
gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2700
ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2760
gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2820
gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2880
ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2940
ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 3000
tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 3060
gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 3120
ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 3180
cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 3240
accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 3300
ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 3360
aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 3420
ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 3480
gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3540
atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3600
ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3660
cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3720
agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3780
gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3840
cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3900
atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3960
cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 4020
ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 4080
ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 4140
aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 4200
tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 4260
ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 4320
ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 4380
ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 4440
gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 4500
cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4560
cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4620
gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4680
ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4740
aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4800
accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4860
gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4920
caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4980
agacccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 5040
tacggcggct tcgtgagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 5100
aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 5160
agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 5220
aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 5280
aagagaatgc tggcctctgc cagattcctg cagaagggaa acgaactggc cctgccctcc 5340
aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 5400
gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 5460
gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 5520
ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5580
cacctgttta ccctgaccaa tctgggagcc cctagagcct tcaagtactt tgacaccacc 5640
atcgaccgga aggtgtacag aagcaccaaa gaggtgctgg acgccaccct gatccaccag 5700
agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 5760
ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa 5820
ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt 5880
ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5940
ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 6000
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 6060
atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 6120
ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 6180
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 6240
gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 6300
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 6360
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 6420
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 6480
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 6540
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 6600
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 6660
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6720
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 6780
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6840
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6900
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6960
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 7020
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 7080
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 7140
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga 7200
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 7260
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 7320
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7380
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7440
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7500
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7560
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7620
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7680
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7740
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7800
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7860
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7920
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7980
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 8040
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 8100
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 8160
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 8220
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 8280
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 8340
gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa 8400
gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 8460
aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 8520
gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 8580
gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8640
ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8700
cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8760
gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c 8811
<210> 2
<211> 4368
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60
tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg actcgcacat tcttcacgtc 120
cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180
tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240
ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300
gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360
cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420
gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480
cgttgaccga atcaccgacc tctctcccca gggggatcca tggtgagcaa gggcgaggag 540
ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 600
ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 660
atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac nctgacctac 720
ggcgtgtagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 780
gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 840
aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 900
ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 960
agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 1020
atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 1080
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 1140
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 1200
gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca 1260
taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 1320
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 1380
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 1440
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagtcgacc gatgcccttg 1500
agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 1560
cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc 1620
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 1680
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 1740
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1800
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1860
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1920
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1980
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2040
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2100
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2160
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2220
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2280
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2340
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2400
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2460
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2520
ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2580
tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 2640
aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc 2700
acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 2760
aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 2820
agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 2880
ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 2940
agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3000
tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3060
tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3120
attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3180
taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3240
aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3300
caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3360
gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3420
cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3480
tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3540
acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 3600
gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 3660
cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 3720
atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 3780
tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 3840
tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 3900
tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 3960
atttaacgcg aattttaaca aaatattaac gcttacaatt tgccattcgc cattcaggct 4020
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct 4080
accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata 4140
tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac 4200
gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 4260
agtgcaggtg ccagaacatt tctctatcga taggtaccga ttagtgaacg gatctcgacg 4320
gtatcgatca cgagactagc cagagatcca ctttggccgc ggctcgag 4368
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
accgagcact acacgccgta ggtc 24
<210> 4
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aaacgaccta cggcgtgtag tgct 24
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
accgcaatcc agacactggt ggtc 24
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aaacgaccac cagtgtctgg attg 24
<210> 7
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accgcgggca gcgaccatag gaag 24
<210> 8
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aaaccttcct atggtcgctg cccg 24
<210> 9
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
accgatggaa agcagacacg atag 24
<210> 10
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aaacctatcg tgtctgcttt ccat 24
<210> 11
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
accggaacat gaactcttac gact 24
<210> 12
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aaacagtcgt aagagttcat gttc 24
<210> 13
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
accgcctcta ttgtgctgtc atgt 24
<210> 14
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aaacacatga cagcacaata gagg 24
<210> 15
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
accgcagcag ctcgtccttc actg 24
<210> 16
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aaaccagtga aggacgagct gctg 24
<210> 17
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
accgtattac agaaaccagc cccg 24
<210> 18
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
aaaccggggc tggtttctgt aata 24
<210> 19
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
accgggctaa cgtgcgggag cgca 24
<210> 20
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aaactgcgct cccgcacgtt agcc 24
<210> 21
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
accgattgat gtaatggatg cagt 24
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
aaacactgca tccattacat caat 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
accggtttca gaatcgaagg gtga 24
<210> 24
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
aaactcaccc ttcgattctg aaac 24
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
accgagacat attcctcact acaa 24
<210> 26
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
aaacttgtag tgaggaatat gtct 24
<210> 27
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
accggcgcat ggccacttcc tgtg 24
<210> 28
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
aaaccacagg aagtggccat gcgc 24
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
accgcttgta tcaggaccac atgc 24
<210> 30
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
aaacgcatgt ggtcctgata caag 24
<210> 31
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
accgaacgtg atggccatgt cgcc 24
<210> 32
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
aaacggcgac atggccatca cgtt 24
<210> 33
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
accgagccag actctgccga tgac 24
<210> 34
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
aaacgtcatc ggcagagtct ggct 24
<210> 35
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
accgtttgga aggaaagtgg tata 24
<210> 36
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
aaactatacc actttccttc caaa 24
<210> 37
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
taggcgggca gcgaccatag gaag 24
<210> 38
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
aaaccttcct atggtcgctg cccg 24
<210> 39
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
taggggctaa cgtgcgggag cgca 24
<210> 40
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
aaactgcgct cccgcacgtt agcc 24
<210> 41
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
taggagacat attcctcact acaa 24
<210> 42
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
aaacttgtag tgaggaatat gtct 24
<210> 43
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
taggtttgga aggaaagtgg tata 24
<210> 44
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
aaactatacc actttccttc caaa 24
<210> 45
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
tctcgcgcgt ttcggtgatg acgg 24
<210> 46
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
aaaaaaagca ccgactcggt gccacttttt c 31
<210> 47
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
accggttcag gttactggag acaa 24
<210> 48
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
aaacttgtct ccagtaacct gaac 24
<210> 49
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
accgctgata cctgaagtgt gtcc 24
<210> 50
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
aaacggacac acttcaggta tcag 24
<210> 51
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
accgcctcag gaggacgacc tggc 24
<210> 52
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
aaacgccagg tcgtcctcct gagg 24
<210> 53
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
accgcactca ccggaacatc acgg 24
<210> 54
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
aaacccgtga tgttccggtg agtg 24
<210> 55
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tagggttcag gttactggag acaa 24
<210> 56
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
aaacttgtct ccagtaacct gaac 24
<210> 57
<211> 166
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 57
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 58
<211> 166
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 58
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 59
<211> 1367
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 59
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp 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 His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1010 1015 1020
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1025 1030 1035 1040
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1045 1050 1055
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val
1060 1065 1070
Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
1075 1080 1085
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1090 1095 1100
Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1105 1110 1115 1120
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Val Ser Pro
1125 1130 1135
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1140 1145 1150
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1155 1160 1165
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1170 1175 1180
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1185 1190 1195 1200
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1205 1210 1215
Arg Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1250 1255 1260
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
1265 1270 1275 1280
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1285 1290 1295
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1300 1305 1310
Thr Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys Tyr Phe Asp Thr
1315 1320 1325
Thr Ile Asp Arg Lys Val Tyr Arg Ser Thr Lys Glu Val Leu Asp Ala
1330 1335 1340
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
1345 1350 1355 1360
Leu Ser Gln Leu Gly Gly Asp
1365
<210> 60
<211> 18
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 60
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val
<210> 61
<211> 1803
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 61
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys
115 120 125
Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His
210 215 220
Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp
225 230 235 240
Glu Arg Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val
245 250 255
Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala
260 265 270
His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn
275 280 285
Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val
290 295 300
Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe
305 310 315 320
Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val
325 330 335
Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile
340 345 350
Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro
355 360 365
Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser
370 375 380
Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser
385 390 395 400
Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp
405 410 415
Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp
420 425 430
Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val
435 440 445
Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala
450 455 460
Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg
465 470 475 480
Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu
485 490 495
Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe
500 505 510
His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu
515 520 525
Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
530 535 540
Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr
545 550 555 560
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile
565 570 575
Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn
580 585 590
Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln
595 600 605
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala
610 615 620
Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile
625 630 635 640
Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile
645 650 655
Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
660 665 670
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp
675 680 685
Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe
690 695 700
Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu
705 710 715 720
Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile
725 730 735
Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu
740 745 750
Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln
755 760 765
Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu
770 775 780
Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr
785 790 795 800
Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln
805 810 815
Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu
820 825 830
Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys
835 840 845
Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr
850 855 860
Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr
865 870 875 880
Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val
885 890 895
Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe
900 905 910
Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu
915 920 925
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val
930 935 940
Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys
945 950 955 960
Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys
965 970 975
Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val
980 985 990
Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr
995 1000 1005
His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu
1010 1015 1020
Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe
1025 1030 1035 1040
Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1045 1050 1055
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly
1060 1065 1070
Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln
1075 1080 1085
Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn
1090 1095 1100
Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu
1105 1110 1115 1120
Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu
1125 1130 1135
His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu
1140 1145 1150
Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His
1155 1160 1165
Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr
1170 1175 1180
Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu
1185 1190 1195 1200
Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu
1205 1210 1215
Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn
1220 1225 1230
Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser
1235 1240 1245
Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp
1250 1255 1260
Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys
1265 1270 1275 1280
Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1285 1290 1295
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp
1300 1305 1310
Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1315 1320 1325
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His
1330 1335 1340
Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1345 1350 1355 1360
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu
1365 1370 1375
Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile
1380 1385 1390
Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly
1395 1400 1405
Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr
1410 1415 1420
Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu
1425 1430 1435 1440
Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
1445 1450 1455
Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg
1460 1465 1470
Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp
1475 1480 1485
Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1490 1495 1500
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser
1505 1510 1515 1520
Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1525 1530 1535
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Val Ser Pro Thr
1540 1545 1550
Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1555 1560 1565
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu
1570 1575 1580
Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1585 1590 1595 1600
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser
1605 1610 1615
Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Arg
1620 1625 1630
Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1635 1640 1645
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu
1650 1655 1660
Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu
1665 1670 1675 1680
Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu
1685 1690 1695
Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg
1700 1705 1710
Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr
1715 1720 1725
Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys Tyr Phe Asp Thr Thr
1730 1735 1740
Ile Asp Arg Lys Val Tyr Arg Ser Thr Lys Glu Val Leu Asp Ala Thr
1745 1750 1755 1760
Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1765 1770 1775
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly
1780 1785 1790
Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1795 1800

Claims (14)

1. a kind of fusion protein, including ecTadA-ecTadA* dimer fragment and SpCas9-NG D10A nickase segment, The ecTadA-ecTadA* dimer fragment includes ecTad segment and ecTadA* segment.
2. fusion protein as described in claim 1, which is characterized in that the amino acid sequence of the ecTadA segment includes:
A) amino acid sequence as shown in SEQ ID NO.57；Or,
B) with amino acid sequence of the SEQ ID NO.57 with 80% or more sequence similarity and with amino acid defined by a) The function of sequence is preferably capable forming dimer with ecTadA* segment and dimer has adenine deaminase activity.
3. fusion protein as described in claim 1, which is characterized in that the amino acid sequence of the ecTadA* segment includes:
C) amino acid sequence as shown in SEQ ID NO.58；Or,
D) with amino acid sequence of the SEQ ID NO.58 with 80% or more sequence similarity and with amino acid defined by c) The function of sequence is preferably capable forming dimer with ecTadA segment and dimer has adenine deaminase activity.
4. fusion protein as described in claim 1, which is characterized in that the ammonia of the SpCas9-NG D10A nickase segment Base acid sequence includes:
E) amino acid sequence as shown in SEQ ID NO.59；Or,
F) with amino acid sequence of the SEQ ID NO.59 with 80% or more sequence similarity and with amino acid defined by e) The function of sequence is preferably capable identification NG as PAM.
5. fusion protein as described in claim 1, which is characterized in that the fusion protein, which is held from 5 ' ends to 3 ', successively includes EcTadA-ecTadA* dimer fragment and SpCas9-NG D10A nickase segment；
And/or it successively includes ecTad segment and ecTadA* that the ecTadA-ecTadA* dimer fragment is held from 5 ' ends to 3 ' Segment；
And/or the fusion protein further includes nuclear localization signal segment, it is preferred that the nuclear localization signal segment is located at 5 ' the ends and/or 3 ' ends of ecTadA-ecTadA* dimer fragment and SpCas9-NG D10A nickase segment, it is preferred that institute The amino acid sequence of nuclear localization signal segment is stated as shown in SEQ ID NO.60.
6. fusion protein as described in claim 1, which is characterized in that the amino acid sequence of the fusion protein such as SEQ ID Shown in No.61.
7. a kind of isolated polynucleotides encode the fusion protein as described in claim 1~6 any claim.
8. a kind of construct, the construct contains the polynucleotides separated as claimed in claim 7.
9. a kind of expression system, the expression system contains to be integrated with outside in construct as claimed in claim 8 or genome The polynucleotides as claimed in claim 7 in source.
10. expression system as claimed in claim 9, which is characterized in that it is thin that the host cell of the expression system is selected from eukaryon Born of the same parents or prokaryotic cell are preferably selected from mouse cell, people's cell, be more preferably selected from mouse brain nerve oncocyte, human embryonic kidney cell, Or human cervical carcinoma cell, it is more preferably selected from N2a cell, HEK293FT cell or Hela cell.
11. fusion protein as described in claim 1~6 any claim, the multicore glycosides separated as claimed in claim 7 Construct sour, as claimed in claim 8 or the expression system as described in claim 9~10 any claim are compiled in gene Purposes in volume.
12. purposes as claimed in claim 11, which is characterized in that the purposes is specially in Eukaryotic gene editing Purposes.
13. a kind of base editor system, including the fusion protein as described in claim 1~6 any claim, the base Editor's system further includes sgRNA.
14. a kind of gene editing method, comprising: by fusion protein as described in claim 1~6 any claim or Base editor's system as claimed in claim 13 carries out gene editing.
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CN110951773A (en) * 2019-12-20 2020-04-03 北京市农林科学院 Application of FNLS-sABE system in creating rice herbicide resistant material
CN110951736A (en) * 2019-12-20 2020-04-03 北京市农林科学院 Nuclear localization signal F4NLS and application thereof in improving base editing efficiency and expanding editable base range
CN110964742A (en) * 2019-12-20 2020-04-07 北京市农林科学院 Preparation method of herbicide-resistant rice
CN110982818A (en) * 2019-12-20 2020-04-10 北京市农林科学院 Application of nuclear localization signal F4NLS in efficient creation of rice herbicide resistant material
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