CN108753778B - 利用碱基编辑修复fbn1t7498c突变的试剂和方法 - Google Patents

利用碱基编辑修复fbn1t7498c突变的试剂和方法 Download PDF

Info

Publication number
CN108753778B
CN108753778B CN201810560722.0A CN201810560722A CN108753778B CN 108753778 B CN108753778 B CN 108753778B CN 201810560722 A CN201810560722 A CN 201810560722A CN 108753778 B CN108753778 B CN 108753778B
Authority
CN
China
Prior art keywords
sgrna
fbn1
mutation
repair
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810560722.0A
Other languages
English (en)
Other versions
CN108753778A (zh
Inventor
黄行许
李广磊
李佳楠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ShanghaiTech University
Original Assignee
ShanghaiTech University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ShanghaiTech University filed Critical ShanghaiTech University
Priority to CN201810560722.0A priority Critical patent/CN108753778B/zh
Priority to PCT/CN2018/096968 priority patent/WO2019227640A1/zh
Priority to EP18889963.7A priority patent/EP3816296A4/en
Priority to US16/470,247 priority patent/US20210198699A1/en
Priority to JP2019528851A priority patent/JP6913965B2/ja
Publication of CN108753778A publication Critical patent/CN108753778A/zh
Application granted granted Critical
Publication of CN108753778B publication Critical patent/CN108753778B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明提供了一种利用碱基编辑修复FBN1T7498C突变的试剂和方法。所述的高效修复FBN1T7498C突变的试剂盒，其特征在于，包括碱基编辑系统以及针对FBN1T7498C位点的修复re‑sgRNA。本发明利用碱基编辑技术，通过精准的CT单碱基突变，从而可以修复FBN1T7498C的突变，从而为治疗该类突变引起的马凡氏综合征提供了高效安全的方法。

Description

利用碱基编辑修复FBN1T7498C突变的试剂和方法
技术领域
本发明涉及基因修复领域，更具体来说，利用碱基编辑修复马凡氏综合征相关的FBN1T7498C突变的方法。
背景技术
目前为止医学上已经鉴定出了接近一万种遗传病，对家庭和社会造成了巨大的负担，然而只有大约6％的遗传病目前可以治疗(Austin and Dawkins,2017)。诊断和治疗遗传病已经是医学上的重要研究内容。高通量测序技术的发展已经使得遗传病的诊断变得较为容易。虽然附植前遗传学诊断(PGD)可以阻断部分遗传病的发生，然而对一些诸如纯合突变等遗传病，尚需要开发更有效的遗传学治疗手段(Dunbar et al.,2018)。
基因编辑技术，尤其是CRISPR/Cas9，已经广泛应用于基因操作，并且可以应用于精准修复致病突变(Komor et al.,2017)。同时目前该技术在人胚胎上的基因编辑也展示出了巨大的优势，提示其在人遗传病治疗中的临床价值(Kang et al.,2016)。然而限于伦理和治疗的效率及脱靶的存在，基因编辑在人胚胎中的应用还有巨大的提升空间(Ruzoand Brivanlou,2017)。CRISPR/Cas9介导的基因编辑是在sgRNA(single guided RNA)通过靶序列互补引导Cas9蛋白定位剪切双链DNA，造成双链DNA断裂(double-strand breaks,DSB)，在没有模板的条件下，发生非同源末端连接(non-homologous end joining,NHEJ)修复，造成移码突变(frameshift mutation)，导致基因敲除(knockout)；在有模板的条件下，通过同源重组进行修复(homology-directed repair,HDR)，实现基因敲入(knockin)，由于HDR效率低(整合很少发生)，而且非同源性末端接合机制容易产生随机插入和删除(indel)，使得在断裂点附近可能随机引入新的碱基，从而导致不精确的基因编辑(Hsu etal.,2014)。此外，CRISPR/Cas9介导的基因编辑总有一些脱靶效应[Gorski et al.,2017]。
最近开发出来的碱基编辑系统，base editor，是在切口酶nCas9的基础上加上了大鼠的胞嘧啶脱氨酶APOBEC1，碱基编辑系统可以在不切割DNA双链的情况下，实现靶位点上C转化为尿嘧啶(Komor et al.,2016)。之后，通过DNA复制或修复，尿嘧啶被转化成胸腺嘧啶(T)，进而实现C到T的转换，类似地，其也能将单碱基G转化成A。由于不切割DNA造成DSB，形成的indel低于1％，实现的基因编辑更精确。碱基编辑系统已经被成功应用于体内碱基编辑，实现了小鼠的CT突变。
马凡氏综合征是一种常染色体显性遗传病，占世界人口的0.2‰，其主要造成人结缔组织的发育异常，目前已有多名知名运动员的猝死和马凡氏综合征有关(Arbustini etal.,2005)。虽然部分患者可以通过手术治疗，但对于后代仍然具有患病的风险，从遗传上治疗引起该病的突变将是根本的方法。前期已有表明FBN1基因的突变是造成马凡氏综合征的主因。因此能否找到高效同时安全性的治疗方法是我们追求的方向。
参考文献
Arbustini,E.,Grasso,M.,Ansaldi,S.,Malattia,C.,Pilotto,A.,Porcu,E.,Disabella,E.,Marziliano,N.,Pisani,A.,Lanzarini,L.,et al.(2005).Identificationof sixty-two novel and twelve known FBN1 mutations in eighty-one unrelatedprobands with Marfan syndrome and other fibrillinopathies.Human mutation 26,494.
Austin,C.P.,and Dawkins,H.J.S.(2017).Medical research:Next decade'sgoals for rare diseases.Nature 548,158.
Dunbar,C.E.,High,K.A.,Joung,J.K.,Kohn,D.B.,Ozawa,K.,and Sadelain,M.(2018).Gene therapy comes of age.Science 359.
Hsu,P.D.,Lander,E.S.,and Zhang,F.(2014).Development and applicationsof CRISPR-Cas9 for genome engineering.Cell 157,1262-1278.
Kang,X.,He,W.,Huang,Y.,Yu,Q.,Chen,Y.,Gao,X.,Sun,X.,and Fan,Y.(2016).Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.Journal of assisted reproduction and genetics 33,581-588.
Komor,A.C.,Badran,A.H.,and Liu,D.R.(2017).CRISPR-Based Technologiesfor the Manipulation of Eukaryotic Genomes.Cell 168,20-36.
Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,and Liu,D.R.(2016).Programmable editing of a target base in genomic DNA without double-strandedDNA cleavage.Nature 533,420-424.
Ruzo,A.,and Brivanlou,A.H.(2017).At Last:Gene Editing in HumanEmbryos to Understand Human Development.Cell Stem Cell 21,564-565.
发明内容
本发明的目的是提供一种高效修复FBN1T7498C突变的试剂盒和方法。
为了达到上述目的，本发明提供了一种高效修复FBN1T7498C突变的试剂盒，其特征在于，包括碱基编辑系统(base editor)以及针对FBN1T7498C位点的修复re-sgRNA。
优选地，所述的碱基编辑系统为BE3，YE1-BE3，YE2-BE3或者YEE-BE3中的一种。
优选地，所述的针对FBN1T7498C位点的修复re-sgRNA的序列为SEQ ID NO.3。
本发明还提供了一种制作突变和修复突变的组合，其特征在于，包括根据FBN1T7498C位点设计突变mt-sgRNA和相应的突变ssODN、针对FBN1T7498C位点的修复re-sgRNA以及碱基编辑系统中的至少一种。
本发明还提供了一种碱基编辑修复突变的方法，其特征在于，包括：在含有FBN1T7498C的突变细胞中，利用针对FBN1T7498C位点的修复re-sgRNA引导碱基编辑系统到突变位点进行碱基编辑修复，收集转染后的细胞。
优选地，所述的含有FBN1T7498C的突变细胞为HEK293T细胞。
优选地，所述的含有FBN1T7498C的突变细胞的构建方法为：根据FBN1T7498C位点设计突变mt-sgRNA和相应的突变ssODN；构建mt-sgRNA的表达载体，体外将Cas9蛋白和转录出来的mt-sgRNA组成RNP结合ssODN的方式并电转HEK293T细胞，流式分选单细胞鉴定出含有FBN1T7498C的突变细胞株。
优选地，所述的针对FBN1T7498C位点的修复re-sgRNA通过根据FBN1T7498C位点设计修复re-sgRNA，并构建U6启动和/或T7启动的表达载体得到。
优选地，所述的mt-sgRNA的序列为SEQ ID NO.1，ssODN的序列为SEQ ID NO.2，re-sgRNA的序列为SEQ ID NO.3。
本发明利用碱基编辑技术在细胞层面进行方法的高效性和安全性证明。
FBN1的突变是造成马凡氏综合征的主要成因，它的发病率在0.2‰左右，从遗传上彻底修复此突变，将是治疗该疾病最有效的措施。碱基编辑系统提供了一种精确改变DNA，即将C变为T的方法。
本发明利用碱基编辑技术，通过精准的CT单碱基突变，从而可以修复FBN1T7498C的突变，从而为治疗该类突变引起的马凡氏综合征提供了高效安全的方法。
附图说明
图1为对患有马凡氏综合征病人的样本进行突变位点的确认。(A)来自血液的样本，(B)来自精液的样本。
图2为利用Cas9/sgRNA结合ssODN的方式在293T细胞中制作突变细胞株。(A)在细胞中制作突变和修复突变的模式图。(B)对FBN1基因设计突变型的sgRNA和ssODN来制作相应突变。(C)转染细胞后对转染效率进行T7EN1酶切鉴定。(D)通过流式分选出22个单细胞克隆，sanger测序确认细胞的突变类型。(E)纯合突变的sanger测序峰图。
图3为对突变的sgRNA进行脱靶检测分选。(A)通过软件分析出突变sgRNA的潜在脱靶位点，对纯合突变细胞株，检测相应的脱靶位点，T7EN1检测脱靶。(B)Sanger测序检测突变。
图4为对突变的细胞株利用碱基编辑进行修复。(A)在突变位点设计相应的修复sgRNA，利用碱基编辑将其修复。(B)细胞转染后的测序检测突变情况。(C)对图B进行TA克隆鉴定修复类型。(D)完全修复后的sanger测序峰图。
图5利用YE1-BE3，YEE-BE3和BE3对突变细胞株修复。
图6为对修复sgRNA进行脱靶检测。(A)利用T7EN1酶切检测脱靶。(B)利用sanger测序确认脱靶情况。
具体实施方式
下面将结合实施例对本发明的实施方案进行清楚、完整的描述，显然，所描述的实施例仅用于说明本发明的一部分实施例，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买获得的常规产品。
以上所述仅为本发明较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
首先，构建不同版本的BE3，即YE1-BE3、YE2-BE3、YEE-BE3和BE3。原始版本BE3购自Addgene(73021)。
1、不同类型APOBEC1的合成。
上述四种BE3只是在rAPOBEC1的编码框上有所差异，YE1-rAPOBEC1(SEQ IDNO.4)、YE2-rAPOBEC1(SEQ ID NO.5)、YEE-rAPOBEC1(SEQ ID NO.6)，其序列分别由生工生物(http://www.sangon.com/)合成。所合成片段的两端分别加有NotI和SmaI的酶切位点。合成的片段克隆到常用的pmd19t载体(TAKARA：6013)上。
2、载体的酶切与纯化
BE3和上述三种合成的载体经过NotI(NEB：R0189L)、SmaI(NEB：R0141L)酶切，体系如下：Buffer(NEB：R0189L)6uL；质粒2ug；NotI 1μL；SmaI 1μL；ddH2O补齐到60μL。混样后于37℃过夜酶切。
酶切产物经过1％的琼脂糖凝胶，进行回收(Axygen：AP-GX-250G)。其中BE3回收大片段的骨架载体，合成载体回收APOBEC1的小片段载体。回收按照试剂盒的使用说明进行(Axygen：AP-PCR-250G)。回收后的片段经过Nanodrop 2000检测浓度。
3、载体的连接、转化与质粒提取。
回收的骨架载体和APOBEC1片段经过连接，连接体系如下：T4连接buffer(NEB：M0202L)1μL，骨架载体20ng，APOBEC1片段50ng，T4连接酶(NEB：M0202L)0.5μL，ddH2O补齐到10μL，16℃连接过夜。转化步骤如下：取20μL感受态细胞(TransGen：CD201)在冰上解冻；2μL的连接产物与感受态细胞混合，冰上放置20分钟；42℃热激60秒；冰上放置2分钟，加入400μL复苏LB培养基(MDBio：L001-1kg)，摇床30分钟；取70μL涂氨苄平板(50μg/ml，37℃培养箱，培养14个小时。
挑选单克隆菌，在4ml液体LB培养基中扩大培养，14小时后提取质粒(Axygene：AP-MN-P-250G)。步骤如下：菌液经过4000转/分钟离心10分钟，倒掉上清培养基；加入350μL的buffer S1，将菌体吹散，转移到2ml离心管中；加入250μL的buffer S2，上下颠倒8次；加入250μL的buffer S3，颠倒混匀6次，产生沉淀；12000转/分钟离心10分钟，取上清过柱；离心1分钟，倒掉废液，加入500μL的W1，离心一分钟，倒掉废液；加入750μL的W2，离心，倒掉上清；加入500μL的W2，离心，倒掉上清；空转1分钟；加入50μL的洗脱液，静置2分钟，离心。获得质粒经过浓度检测，取10μL送测序，阳性质粒保存在-20℃。
最终构建成如下四种BE3：
(1)YE1-BE3，SEQ ID NO.7；
(2)YE2-BE3，SEQ ID NO.8；
(3)YEE-BE3，SEQ ID NO.9；
(4)BE3，SEQ ID NO.10。
在下面进行突变位点修复时，可以采用上述任一种BE3，优选为(1)或(4)。
实施例1
本实施例中，在细胞株上利用Cas9/sgRNA结合ssODN制作突变FBN1T7498C突变细胞株，并利用碱基编辑系统对突变株进行修复(图2)。
1.1质粒构建
在突变位点附近，设计突变mt-sgRNA(SEQ ID NO.1)，合成oligos，上游序列为：5’-taggCGCCAATGGTGTTAACACAT-3’(SEQ ID NO.(14))，下游序列为：5’-aaacATGTGTTAACACCATTGGCG-3’(SEQ ID NO.(15))，上下游序列通过程序(95℃，5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的PUC57-T7sgRNA载体(addgene：51132)上。线性化体系如下所示：PUC57-T7sgRNA 2μg；buffer(NEB：R0539L)6μL；BsaI 2μL；ddH2O补齐到60μL。37℃酶切过夜。所使用的同源模板ssODN(SEQ ID NO.2)，利用PAGE纯化的方式由生工生物公司(http://www.sangon.com/)合成。同时在突变位点附近，依据碱基编辑作用的特点，设计修复re-sgRNA(SEQ ID NO.3)，合成oligos，上游序列为5’-accgCTACGTGTTAACACCATTGG-3’(SEQ IDNO.16)，下游序列为5’-aaacCCAATGGTGTTAACACGTAG-3’(SEQ ID NO.17)。上下游序列通过程序(95℃，5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的PGL3-U6sgRNA载体上。同时合成上游引物：5’-taggCTACGTGTTAACACCATTGG-3”(SEQ ID NO.18)和下游引物：5’-aaacCCAATGGTGTTAACACGTAG-3’(SEQ ID NO.19)，经过退火连接到线性化的PUC57-T7sgRNA载体上。退火程序、线性化体系与程序如上。连接体系如下：T4连接buffer(NEB：M0202L)1μL，线性化载体20ng，退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH2O补齐到10μL.16℃连接过夜。连接的载体通过转化，挑菌，鉴定，鉴定引物为U6载体为上游序列：5’-TTTCCCATGATTCCTTCATA-3’(SEQ ID NO.20)，下游序列为相应oligo的下游序列。T7载体为上游序列：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’(SEQ ID NO.21)，下游序列为相应oligo的下游序列。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)测定浓度备用。获得的突变质粒命名为mt-T7-sgRNA(SEQ ID NO.11)、re-U6-sgRNA(SEQ ID NO.12)和re-T7-sgRNA(SEQ ID NO.13)。
1.2 sgRNA的体外转录
以构建的PUC57-T7sgRNA为模板，扩增含有sgRNA的片段，所用引物为：F:5’-TCTCGCGCGTTTCGGTGATGACGG-3’,(SEQ ID NO.22)R:5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3’(SEQ ID NO.23)。扩增体系如下：2Xbuffer(诺唯赞：P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；模板1ng；DNA聚合酶(诺唯赞：P505)0.5μL；ddH2O补齐到50μL。扩增出来的PCR产物经过下述步骤纯化：每100μL体积加4μL RNAsecure(Life：AM7005)；60℃15分钟；加入三倍体积的PCR-A(Axygen：AP-PCR-250G)过柱，离心，12000转/分钟离心1分钟；加入500μL W2，离心1分钟；空转1分钟；加入20μL无RNAase水洗脱。
利用体外转录试剂盒(Ambion,Life Technologies,AM1354)转录，步骤如下：反应体系为：reaction buffer 1μL；enzyme mix 1μL；A 1μL；T 1μL；G 1μL；C 1μL；模板800ng；H2O补齐到10μL。上述体系混匀后37℃反应5个小时。加入1μLDNase，37℃反应15分钟。利用回收试剂盒(Ambion,Life Technologies,AM1908)回收转录的sgRNA，步骤如下：上步反应体积加入90μL Elution solution移植1.5mlEP管；加入350μL Binding solution混匀；加入250μL无水乙醇混匀；上柱；10000转/分钟离心30秒，倒掉废液；加入500μL Washingsolution，10000转/分钟离心30秒，倒掉废液；空转1分钟；换收集管，加入100μL Elutionsolution洗脱；加入10μL醋酸铵(Ambion,Life Technologies,AM1908)混匀；加入275μL无水乙醇混匀；-20℃放置30分钟，同时准备70％乙醇放置-20℃；4℃环境下13000转/分钟离心15分钟。弃上清，加入500μL 70％乙醇；离心5分钟，吸走废液，晾干5分钟；加入20μL的水溶解；取1μL测浓度。
1.3细胞的培养与电转
(1)以HEK293T细胞(购自ATCC)为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)转染前两个小时换成无抗生素的培养基，利用LONZA转染试剂(SFKIT)按照说明书转染，细胞通过计数得1X106个。将Cas9(Sigma：ESPCAS9PRO-50UG)，sgRNA和ssODN按照3μg，1.5μg和3μg的质量混合。电转程序采用DS150，电转后的细胞在6cm的平皿中培养两天。
(3)细胞通过流式分选仪，分选单细胞培养，等两周以后，通过裂解鉴定基因型，裂解液的成分为50mM KCl,1.5mM MgCl2,10mM Tris pH 8.0,0.5％Nonidet P-40,0.5％Tween 20,100μg/ml protease K。挑选纯合突变的细胞株扩大培养。
(4)对突变的细胞株，通过http://crispr.mit.edu/,https://crispr.cos.uni-heidelberg.de/鉴定出7个相关的脱靶位点，如表1所示。
(5)通过设计相应的引物对脱靶位点进行鉴定，引物序列如表3所示。扩增的PCR产物经过T7EN1和测序鉴定，并没有发现脱靶(图3)。
1.4突变细胞株的修复(图4，图5)
(1)本发明对突变细胞株进行培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前分至6孔板中，待密度达到70％-80％时进行转染。
(3)转染以脂质体转染为例。按照LipofectamineTM 2000 Transfection Reagent(Invitrogen,11668-019)的操作手册，将2μg BE3或YE1-BE3或YEE-BE3质粒与1μg re-sgRNApGL3-U6-sgRNA质粒混匀,共转染至每孔细胞中，6-8小时后换液，72小时后收取细胞。
(4)对收集到的细胞首先进行PCR产物测序，发现存在修复的峰值(图3)。进一步得，对PCR产物进行TA克隆，在挑选出来的克隆里面，修复的克隆效率有50％。
(5)我们对修复的re-sgRNA进行脱靶分析，同样，我们找到了8个潜在的脱靶位点，如表2和图3所示，同样，我们对潜在脱靶位点进行了分析和检测(表3，图6)，同样，没有发现脱靶现象。
表1 mt-sgRNA潜在的脱靶位点
Figure GDA0003003084120000091
表2 re-sgRNA潜在的脱靶位点
Figure GDA0003003084120000092
表3本项目所使用的引物序列
Figure GDA0003003084120000093
Figure GDA0003003084120000101
Figure GDA0003003084120000111
序列表
<110> 上海科技大学
<120> 利用碱基编辑修复FBN1T7498C突变的试剂和方法
<160> 57
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 1
cgccaatggt gttaacacat agg 23
<210> 2
<211> 110
<212> DNA
<213> Artificial Sequence
<400> 2
gacgtatggt gttgggtaaa tccgggagga catttgcatg tgaagccgcc aatggtgtta 60
acacgtagga actggcagtt gtgttgcttg gttgcacact catcaagatc 110
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 3
ccgccaatgg tgttaacacg tag 23
<210> 4
<211> 687
<212> DNA
<213> Artificial Sequence
<400> 4
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctac agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg accccgagaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaa 687
<210> 5
<211> 687
<212> DNA
<213> Artificial Sequence
<400> 5
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctat agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctggaagatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaa 687
<210> 6
<211> 687
<212> DNA
<213> Artificial Sequence
<400> 6
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctac agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg accccgagaa tcgacaaggc ctggaggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaa 687
<210> 7
<211> 8532
<212> DNA
<213> Artificial Sequence
<400> 7
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 gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctacag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
cccgagaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820
ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940
tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040
acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100
gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160
agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220
agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520
atcaagtgta tc 8532
<210> 8
<211> 8532
<212> DNA
<213> Artificial Sequence
<400> 8
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 gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctatag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct ggaagatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820
ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940
tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040
acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100
gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160
agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220
agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520
atcaagtgta tc 8532
<210> 9
<211> 8604
<212> DNA
<213> Artificial Sequence
<400> 9
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 atagggagac ccaagctggc tagcaccatg 420
ggacctaaga aaaagaggaa ggtgtctaga gactacaagg atgacgacga taaaggatcc 480
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 540
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 600
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 660
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 720
aggtgcagca ttacctggtt tctcagctac agcccatgcg gcgaatgtag tagggccatc 780
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 840
caccacgctg accccgagaa tcgacaaggc ctggaggatt tgatctcttc aggtgtgact 900
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 960
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 1020
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 1080
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 1140
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 1200
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 1260
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 1320
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 1380
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 1440
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1500
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1560
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1620
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1680
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1740
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1800
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1860
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1920
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1980
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 2040
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 2100
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 2160
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 2220
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 2280
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 2340
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 2400
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 2460
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2520
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2580
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg 2640
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2700
aactttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2760
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2820
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2880
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2940
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 3000
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 3060
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 3120
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 3180
acgggctggg gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 3240
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg 3300
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 3360
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 3420
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3480
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3540
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3600
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3660
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3720
gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3780
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3840
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3900
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3960
attaaacgtc agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat 4020
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 4080
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 4140
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 4200
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 4260
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 4320
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 4380
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 4440
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4500
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4560
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4620
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4680
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4740
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4800
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4860
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4920
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4980
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 5040
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 5100
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 5160
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 5220
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 5280
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 5340
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 5400
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 5460
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5520
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5580
ggtggttctc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat caccattgag 5640
tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5700
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5760
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5820
ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 5880
gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct 5940
agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 6000
ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga 6060
gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 6120
gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 6180
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 6240
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 6300
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 6360
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 6420
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 6480
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 6540
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 6600
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 6660
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 6720
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 6780
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 6840
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 6900
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 6960
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 7020
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 7080
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 7140
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 7200
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 7260
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 7320
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 7380
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 7440
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 7500
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 7560
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 7620
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 7680
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 7740
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 7800
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 7860
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 7920
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 7980
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 8040
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 8100
aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc 8160
tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 8220
tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 8280
acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 8340
ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 8400
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 8460
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 8520
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 8580
acttggcagt acatcaagtg tatc 8604
<210> 10
<211> 8532
<212> DNA
<213> Artificial Sequence
<400> 10
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctacag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
cccgagaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820
ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940
tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040
acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100
gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160
agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220
agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520
atcaagtgta tc 8532
<210> 11
<211> 2791
<212> DNA
<213> Artificial Sequence
<400> 11
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc ctaatacgac tcactatagg cgccaatggt gttaacacat 480
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 540
ggcaccgagt cggtgctttt tttaaagggc ccgtcgactg cagaggcctg catgcaagct 600
tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 660
acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 720
tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 780
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgcggccgcc 840
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 900
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 960
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1020
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1080
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1140
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1200
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1260
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1320
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1380
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1440
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1500
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1560
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1620
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1680
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1740
atctaaagta tatatgagta aacttggtct gacagttaga aaaactcatc gagcatcaaa 1800
tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 1860
tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 1920
tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 1980
aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt 2040
ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 2100
ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 2160
tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc 2220
agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 2280
ttcccaggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 2340
atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 2400
tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 2460
tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 2520
tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga 2580
atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2640
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2700
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 2760
aaaaataggc gtatcacgag gccctttcgt c 2791
<210> 12
<211> 4950
<212> DNA
<213> Artificial Sequence
<400> 12
ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60
cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300
tgtggaaagg acgaaacacc gctacgtgtt aacaccattg ggttttagag ctagaaatag 360
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 420
ttttaaagaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa 480
aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat 540
acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag 600
ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag 660
gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg 720
gcgccgaccc tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg 780
ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt 840
ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc 900
tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca 960
atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg 1020
cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca 1080
agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc 1140
cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca 1200
cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg 1260
ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac 1320
tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg 1380
cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg 1440
gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc 1500
tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc 1560
ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg 1620
agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg 1680
agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt 1740
gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa 1800
ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag 1860
atcttagcca ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac 1920
attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 1980
atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 2040
aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc 2100
aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag 2160
agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact 2220
tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt 2280
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2340
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2400
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 2460
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2520
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2580
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 2640
tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 2700
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 2760
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 2820
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 2880
gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2940
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3000
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3060
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 3120
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3180
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3240
tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3300
ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac 3360
gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 3420
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 3480
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 3540
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 3600
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 3660
tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 3720
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3780
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 3840
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 3900
aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 3960
actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4020
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4080
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4140
aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4200
ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4260
ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4320
ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4380
ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4440
ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4500
gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4560
tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4620
ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc ccattcgcca ttcaggctgc 4680
gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac 4740
catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc 4800
tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc 4860
tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag 4920
tgcaggtgcc agaacatttc tctatcgata 4950
<210> 13
<211> 2791
<212> DNA
<213> Artificial Sequence
<400> 13
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc ctaatacgac tcactatagg ctacgtgtta acaccattgg 480
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 540
ggcaccgagt cggtgctttt tttaaagggc ccgtcgactg cagaggcctg catgcaagct 600
tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 660
acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 720
tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 780
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgcggccgcc 840
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 900
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 960
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1020
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1080
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1140
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1200
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1260
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1320
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1380
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1440
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1500
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1560
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1620
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1680
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1740
atctaaagta tatatgagta aacttggtct gacagttaga aaaactcatc gagcatcaaa 1800
tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 1860
tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 1920
tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 1980
aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt 2040
ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 2100
ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 2160
tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc 2220
agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 2280
ttcccaggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 2340
atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 2400
tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 2460
tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 2520
tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga 2580
atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2640
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2700
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 2760
aaaaataggc gtatcacgag gccctttcgt c 2791
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 14
taggcgccaa tggtgttaac acat 24
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 15
aaacatgtgt taacaccatt ggcg 24
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 16
accgctacgt gttaacacca ttgg 24
<210> 17
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 17
aaacccaatg gtgttaacac gtag 24
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 18
taggctacgt gttaacacca ttgg 24
<210> 19
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 19
aaacccaatg gtgttaacac gtag 24
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 20
tttcccatga ttccttcata 20
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 21
cgccagggtt ttcccagtca cgac 24
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 22
tctcgcgcgt ttcggtgatg acgg 24
<210> 23
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 23
aaaaaaagca ccgactcggt gccacttttt c 31
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 24
actcaccaat gcaggacgta 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 25
agctgcttca tagggtcagc 20
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 26
gctgaagtct ccacccacc 19
<210> 27
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 27
tgtctctcct tgccttttg 19
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
tcaagggaca ggagtaggca 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 29
ttggggcagg aggttttgtt 20
<210> 30
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 30
atcttaatca gggccttga 19
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 31
gccttcattc catcaactg 19
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 32
caggttcgtg tcgcagtagc 20
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 33
ctgtgttgcc agcacgaaa 19
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 34
tggtagtggt tggtgacact 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 35
cgttacattg ggaagcggaa 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 36
ggattcaaca tagattggaa 20
<210> 37
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 37
cccgtttaca cattgcta 18
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 38
ttctagtagg tgaaaaaggg 20
<210> 39
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 39
ttggacacca catagacag 19
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 40
tattattgct aaaccgaaac ca 22
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 41
agcccctcac ccactcat 18
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 42
agaggcttgc gaaggacatc 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 43
atttggtcta gggcagaggc 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 44
attattcaca agttatggta 20
<210> 45
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 45
taaccctctt ctttgtaa 18
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 46
aagggactgt ttttgtcctg tca 23
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 47
gtgaaaccac catgacatga agt 23
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 48
gtcatacttg gccagggtcc 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 49
cccacgtgag ctggctaaaa 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 50
tgatcagcat gtggagcctg 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 51
gaagtcagcc aggagccatt 20
<210> 52
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 52
gagttaggag tgggaagg 18
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 53
acaaaggaca gtaatgaaga g 21
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 54
tttgcctcct tgattccccc 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 55
gtggatggtg tggaggtgag 20
<210> 56
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 56
cgataaaggg atcagtcact aa 22
<210> 57
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 57
gctccaggtc cacaaacac 19

Claims (7)

1.一种高效修复FBN1T7498C突变的试剂盒，其特征在于，包括碱基编辑系统以及针对FBN1T7498C位点的修复re-sgRNA；所述的针对FBN1T7498C位点的修复re-sgRNA的序列为SEQ IDNO.3。
2.如权利要求1所述的高效修复FBN1T7498C突变的试剂盒，其特征在于，所述的碱基编辑系统为BE3，YE1-BE3，YE2-BE3或者YEE-BE3中的一种。
3.一种制作突变和修复突变的组合，其特征在于，包括根据FBN1T7498C位点设计突变mt-sgRNA和相应的突变ssODN、针对FBN1T7498C位点的修复re-sgRNA以及碱基编辑系统；所述的mt-sgRNA的序列为SEQ ID NO.1，ssODN的序列为SEQ ID NO.2，re-sgRNA的序列为SEQ IDNO.3。
4.一种非治疗目的的碱基编辑修复突变的方法，其特征在于，包括：在含有FBN1T7498C的突变细胞中，利用针对FBN1T7498C位点的修复re-sgRNA引导碱基编辑系统到突变位点进行碱基编辑修复，收集转染后的细胞；所述re-sgRNA的序列为SEQ ID NO.3。
5.如权利要求4所述的碱基编辑修复突变的方法，其特征在于，所述的含有FBN1T7498C的突变细胞为HEK293T细胞。
6.如权利要求4所述的碱基编辑修复突变的方法，其特征在于，所述的含有FBN1T7498C的突变细胞的构建方法为：根据FBN1T7498C位点设计突变mt-sgRNA和相应的突变ssODN；构建mt-sgRNA的表达载体，体外将Cas9蛋白和转录出来的mt-sgRNA组成RNP结合ssODN的方式并电转HEK293T细胞，流式分选单细胞鉴定出含有FBN1T7498C的突变细胞株；所述的mt-sgRNA的序列为SEQ ID NO.1，ssODN的序列为SEQ ID NO.2。
7.如权利要求4所述的碱基编辑修复突变的方法，其特征在于，所述的针对FBN1T7498C位点的修复re-sgRNA通过根据FBN1T7498C位点设计修复re-sgRNA，并构建U6启动和/或T7启动的表达载体得到。
CN201810560722.0A 2018-06-01 2018-06-01 利用碱基编辑修复fbn1t7498c突变的试剂和方法 Active CN108753778B (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CN201810560722.0A CN108753778B (zh) 2018-06-01 2018-06-01 利用碱基编辑修复fbn1t7498c突变的试剂和方法
PCT/CN2018/096968 WO2019227640A1 (zh) 2018-06-01 2018-07-25 利用碱基编辑修复fbn1t7498c突变的试剂和方法
EP18889963.7A EP3816296A4 (en) 2018-06-01 2018-07-25 REAGENT AND METHOD FOR REPAIRING THE FBN1T7498C MUTATION USING BASE EDIT
US16/470,247 US20210198699A1 (en) 2018-06-01 2018-07-25 Kit for reparing fbn1t7498c mutation, combination for making and repairing mutation, and method of repairing thereof
JP2019528851A JP6913965B2 (ja) 2018-06-01 2018-07-25 Ｆｂｎ１ｔ７４９８ｃ突然変異を修復するキット、ｆｂｎ１ｔ７４９８ｃ突然変異の作製及び修復方法、塩基編集によるｆｂｎ１ｔ７４９８ｃ突然変異の修復方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810560722.0A CN108753778B (zh) 2018-06-01 2018-06-01 利用碱基编辑修复fbn1t7498c突变的试剂和方法

Publications (2)

Publication Number Publication Date
CN108753778A CN108753778A (zh) 2018-11-06
CN108753778B true CN108753778B (zh) 2021-11-02

Family

ID=64002143

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810560722.0A Active CN108753778B (zh) 2018-06-01 2018-06-01 利用碱基编辑修复fbn1t7498c突变的试剂和方法

Country Status (5)

Country Link
US (1) US20210198699A1 (zh)
EP (1) EP3816296A4 (zh)
JP (1) JP6913965B2 (zh)
CN (1) CN108753778B (zh)
WO (1) WO2019227640A1 (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666678A (zh) * 2019-01-29 2019-04-23 四川省人民医院 检测马凡综合征的试剂盒
CN109666676A (zh) * 2019-01-29 2019-04-23 四川省人民医院 筛查马凡综合征的试剂盒
CN109666729A (zh) * 2019-01-29 2019-04-23 四川省人民医院 一种马凡综合征筛查试剂盒
CN109666673B (zh) * 2019-02-01 2022-04-08 国家卫生计生委科学技术研究所 利用碱基编辑修复与胆固醇酯贮积症相关的e8sjm-1g>a突变的试剂和方法
CN109762846B (zh) * 2019-02-01 2020-11-24 国家卫生健康委科学技术研究所 利用碱基编辑修复与克拉伯病相关的galcc1586t突变的试剂和方法
CN114107452B (zh) * 2021-12-07 2022-06-24 深圳市眼科医院 一种基于fbn1基因插入突变的马凡综合征检测试剂盒

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104120132A (zh) * 2013-04-28 2014-10-29 福建省立医院 Fbn1基因突变体及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3310932B1 (en) * 2015-06-17 2023-08-30 The UAB Research Foundation Crispr/cas9 complex for genomic editing
AR110075A1 (es) * 2016-11-14 2019-02-20 Inst Genetics & Developmental Biology Cas Un método para edición basal en plantas
CN106916852B (zh) * 2017-04-13 2020-12-04 上海科技大学 一种碱基编辑系统及其构建和应用方法
CN107384920B (zh) * 2017-05-10 2020-07-14 中山大学 一套基于化脓性链球菌的碱基编辑系统及其在基因编辑中的应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104120132A (zh) * 2013-04-28 2014-10-29 福建省立医院 Fbn1基因突变体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer;Massimo Caputi et al;《Genes & Development》;20021231;第16卷;第1754-1759页 *
Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos;Yanting Zeng et al;《Molecular Therapy》;20180813;第26卷(第11期);第2631-2637页 *

Also Published As

Publication number Publication date
CN108753778A (zh) 2018-11-06
JP6913965B2 (ja) 2021-08-04
EP3816296A4 (en) 2022-10-12
WO2019227640A1 (zh) 2019-12-05
JP2020532277A (ja) 2020-11-12
EP3816296A1 (en) 2021-05-05
US20210198699A1 (en) 2021-07-01

Similar Documents

Publication Publication Date Title
CN108753778B (zh) 利用碱基编辑修复fbn1t7498c突变的试剂和方法
CN106916852B (zh) 一种碱基编辑系统及其构建和应用方法
CN111763686B (zh) 实现c到a以及c到g碱基突变的碱基编辑系统及其应用
CN101935663B (zh) 调控花青素合成与代谢的小麦新基因TaMYB3
US20220056475A1 (en) Recombinant poxviruses for cancer immunotherapy
CN108138121B (zh) 用微生物高水平生产长链二羧酸
CN108026523A (zh) 向导rna组装载体
CN107090441A (zh) 用于获得高产量重组蛋白表达的基于mgmt的方法
KR20150042856A (ko) 클라빈-유형 알칼로이드의 생산을 위한 유전자 및 방법
CN108779480A (zh) 生产鞘氨醇碱和鞘脂类的方法
CN111088176B (zh) 产β-胡萝卜素的基因工程菌及其应用
CN115927299A (zh) 增加双链rna产生的方法和组合物
RU2730667C2 (ru) Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген, выбранный из группы генов KRT5, KRT14, LAMB3, COL7A1, для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli SCS110-AF/VTvaf17-KRT5, или Escherichia coli SCS110-AF/VTvaf17-KRT14, или Escherichia coli SCS110-AF/VTvaf17-LAMB3, или Escherichia coli SCS110-AF/VTvaf17-COL7A1, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора
KR102178022B1 (ko) Fgf21 반응성 리포터 유전자 세포주
CN109762846B (zh) 利用碱基编辑修复与克拉伯病相关的galcc1586t突变的试剂和方法
US6531289B1 (en) Regulated gene expression in yeast and method of use
CN110734480B (zh) 大肠杆菌分子伴侣GroEL/ES在协助合成植物Rubisco中的应用
CN113061626B (zh) 一种组织特异性敲除斑马鱼基因的方法及应用
CN111534542A (zh) piggyBac转座子系统介导的真核生物转基因细胞系及构建方法
CN113039278A (zh) 通过指导的内切核酸酶和单链寡核苷酸进行基因组编辑
CN116549630A (zh) 一种由腺相关病毒载体介导表达的抗血管内皮生长因子基因药物及其制备方法和应用
CN116135974A (zh) 一种重组糖基化酶碱基编辑系统及其应用
CN114438083A (zh) 识别猪PERV基因的sgRNA及其编码DNA和应用
CN101652482A (zh) 由代谢工程化酵母进行的丁醇生产
US6716601B1 (en) Compositions and methods utilizing the yeast ZE01 promoter

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant