基于CjCas9和VPR核心结构域的融合蛋白、相应的DNA靶向激活系统及其应用的制作方法

本发明属于生物技术领域，涉及一种基于cjcas9及其突变体和vpr核心结构域的融合蛋白、相应的dna靶向激活系统及其应用。

背景技术：

crispr/cas9系统是目前应用广泛的基因编辑系统，是从细菌和古细菌降解外源dna的适应性免疫机制改造而来。crispr/cas9系统主要包含两个元件：cas9蛋白和向导rna。cas9蛋白和向导rna结合后会在向导rna的引导下对特定位点dna双链进行切割，从而产生断裂，这比之前的基因编辑方法要简单得多。而近年来，crispr/cas9系统也逐渐应用于基因调节领域。利用失去切割活性的crispr/cas9系统并结合转录激活或者抑制因子，也可以在真核以及原核生物激活或抑制基因的表达，这些使crispr系统可能成为下一基因治疗的有利平台。

cjcas9来源于空肠弯曲菌，与其他crispr系统相比，它具有以下优势：首先，pam区不同。crispr系统对dna的靶向受到pam区的限制，cjcas9的pam区为nnnvryac,不同于spcas9识别g-richpam，cpf1复合物识别的t-richpam序列,这为crispr系统的编辑位点提供了更多的选择。其次，特异性不同。相对于spcas9具有较高的脱靶率，cjcas9具有更高的特异性。更重要的是，大小不同。cjcas9是目前已知的最小的cas9蛋白，相比于spcas9有1368个氨基酸，cjcas9只有984个氨基酸，更容易进入组织和细胞，这极大地拓展了crispr系统在体内外的应用。

vpr是三种转录激活因子vp64、p65和rta的核心结构域组成的融合蛋白。一方面，它具有这三种转录激活因子的协同作用，使其能在更多的位点实现更好的激活效果。另外一方面，它是由这三种转录激活因子的核心结构域组成的融合蛋白，长度仅325个氨基酸，提高了它的应用范围。

目前，crispr/cas9系统对基因的调控主要应用于体外，而在体内应用较少，其原因有很多，其中最重要的是大部分cas9蛋白较大，超过了一些包装方法如aav病毒的包装范围，再结合转录调节系统，更是超过了大部分包装系统的容量，使得调节系统很难传递到体内的组织细胞发挥作用。同时基因调节的效果以及系统的特异性也是其局限因素。在本发明中，将目前体积最小的cjcas9蛋白和三种转录激活因子vp64、p65和rta的核心结构域组成的融合蛋白vpr结合，整个激活系统具有小体积、强激活效果以及高特异性。可以通过一些常用的包装方法如aav病毒传递至体内，发挥其激活效果。

技术实现要素：

本发明目的在于：针对已有基因激活方法靶向性低，激活效率低，以及系统体积过大难以应用于体内等问题，将目前体积最小的crispr/cas9系统蛋白cjcas9和转录激活因子vpr结合，提供一种体积小、激活效果强以及高特异性的激活系统，可以实现体内外高效的基因激活。

为了实现上述发明目的，本发明提供了一种融合蛋白，包括两个异源多肽结构域，其中一个多肽结构域包含具有转录激活活性的vpr蛋白，所述vpr蛋白由三种转录激活因子vp64、p65、rta的核心结构域组成；另一种多肽结构域包含cjcas9蛋白，所述cjcas9蛋白为dcjcas9亚型、mini-dcjcas9亚型或cjcas9野生型。所述的cjcas9野生型为目前现有的亚型，序列如seqidno：3中倾斜字体所示；所述dcjcas9亚型是本发明基于cjcas9野生型在d8a和h559a单位点氨基酸突变，使cjcas9野生型无酶切割活性有靶向基因识别特性，序列如seqidno：1中倾斜字体所示；所述mini-dcjcas9亚型是本发明基于cjcas9野生型在d8a单位点氨基酸突变以及大部分hnh结构域(△495-601氨基酸)的缺失，序列如seqidno：2中倾斜字体所示。(除了野生型，其他都是之前没有的)。

进一步地，所述vpr与dcjcas9亚型的融合蛋白，其氨基酸序列如seqidno：1所示，其核苷酸序列如seqidno：5所示；所述vpr与mini-dcjcas9亚型的融合蛋白，其氨基酸序列如seqidno：2所示，其核苷酸序列如seqidno：6所示；所述vpr与cjcas9野生型的融合蛋白，其氨基酸序列如seqidno：3所示，其核苷酸序列如seqidno：7所示。

进一步地，本发明基于所述vpr与mini-dcjcas9亚型的融合蛋白，将转录因子vpr中的vp64进行密码子优化，vpr整体缩小27个氨基酸序列，vpr与minidcjcas9之间的linker缩小33个氨基酸，其氨基酸序列如seqidno：4所示，其核苷酸序列如seqidno：8所示。

seqidno：1

vpr-dcjcas9氨基酸序列(正常字体为vpr；加粗字体为linker；倾斜字体为dcjcas9)：

seqidno：2

vpr-minidcjcas9氨基酸序列(正常字体为vpr；加粗字体为linker；倾斜字体为minidcjcas9)：

seqidno：3

vpr-wtcjcas9氨基酸序列(正常字体为vpr；加粗字体为linker；倾斜字体为wtcjcas9)：

seqidno：4

vpr-s-linker-minidcjcas9-d2vp64new氨基酸序列(正常字体为vpr，密码子优化及删减；加粗字体为linker，已删减；倾斜字体为minidcjcas9)：

seqidno：5

vpr-dcjcas9dna序列(正常字体为vpr；加粗字体为linker；倾斜字体为dcjcas9)：

seqidno：6

vpr-minidcjcas9dna序列(正常字体为vpr；加粗字体为linker；倾斜字体为minidcjcas9)：

seqidno：7

vpr-wtcjcas9dna序列(正常字体为vpr；加粗字体为linker；倾斜字体为wtcjcas9)：

seqidno：8

vpr-s-linker-minidcjcas9-d2vp64new序列(正常字体为vpr，密码子优化及删减；加粗字体为linker，已删减；倾斜字体为minidcjcas9)：

一种表达载体，该表达载体用于表达上述所述的融合蛋白。

进一步地，所述表达载体的启动子为cmv，入核信号为sv40nls，vpr蛋白和cjcas9蛋白之间插入一段连接序列linker，序列如seqidno：1中加粗字体所示。

一种dna靶向激活系统，包括上述所述的融合蛋白和至少一种向导rna，所述向导rna包含针对目的基因启动子区域设计的一段长度为14bp-22bp的序列和一段80bp的骨架序列。

进一步地，vpr与dcjcas9亚型、mini-dcjcas9亚型或cjcas9野生型的融合蛋白均可实现基因的激活，其中vpr与dcjcas9亚型的融合蛋白具有相对更高的激活效率；vpr与mini-dcjcas9亚型的融合蛋白更小，在动物体内应用中具有更好的前景；vpr与cjcas9野生型融合蛋白可利用15bp和22bp的导向grna，同时进行基因激活和切割；同时，本发明还基于vpr与mini-dcjcas9亚型的融合蛋白，将转录因子vpr中的vp64进行密码子优化，vpr整体缩小27个氨基酸序列，vpr与minidcjcas9之间的linker缩小33个氨基酸，使得整个激活系统更小，融合蛋白的表达量更强。

进一步地，所述cjcas9的向导rna骨架序列包含两种，一种是已知的骨架序列wt向导rna骨架，其核苷酸序列如seqidno：9所示；另一种是含有t5a和a24t单位点碱基突变的f向导rna骨架，其核苷酸序列如seqidno：10所示。

seqidno：9

cjcas9向导rna的wt骨架序列：

gttttagtccctgaaaagggactaaaataaagagtttgcgggactctgcggggttacaatcccctaaaaccgcttttttt

seqidno：10

cjcas9向导rna的f骨架序列(加粗倾斜的字体为突变点)：

进一步地，所述cjcas9的向导rna骨架序列是含有t5a和a24t单位点碱基突变的f向导rna骨架。

现有技术中只有利用野生型cjcas9加22bp导向rna做基因切割，没有人利用cjcas9做过激活。本申请1、把野生型cjcas9突变为dcjcas9，然后与之前有的vpr结合用于激活；2、把野生型cjcas9突变并缩短为mini-dcjcas9，也可以实现激活，同时因为删减质粒更小，体内应用范围更广；3、直接利用野生型cjcas9，发现结合15bp的导向rna，也可以实现激活，而且还可以结合靶向其他基因22bp的导向，可以实现对一个基因激活的同时实现对其他基因的切割；4、将vpr与mini-dcjcas9的融合蛋白进一步缩减优化，vpr缩小，linker缩小，整个蛋白更小，同时因为对vp64密码子优化，使得这个融合蛋白翻译效率更高，表达量更强；5、向导rna骨架序列：wt向导rna骨架是之前有的，将t5a和a24t单位点碱基突变的f向导rna骨架是本申请改进的，改进之后激活效果更好。

上述所述的dna靶向激活系统在进行基因的靶向激活中的应用。

进一步地，所述靶向基因为活体动物基因。本发明提供的dna靶向激活系统可以高效激活基因的表达，具体包括如下步骤：(1)构建cjcas9和vpr核心结构域的融合蛋白表达载体；(2)构建表达特定向导rna的载体；(3)将步骤(1)所述融合蛋白表达载体和步骤(2)所述表达特定向导rna的载体混合，通过脂质体转染试剂聚乙烯亚胺(pei)共转染到哺乳动物细胞，在细胞内表达的融合蛋白与其向导rna结合，将转录激活因子vpr靶向目标基因区；(4)48h后提取mrna，检查目标基因mrna表达水平；再通过全转录水平mrna测序，分析靶向激活结果和特异性水平。

与现有技术相比，本发明的优势在于：本发明提供的dna靶向激活系统具有体积小特异性高，激活效果强易于合成，成本低等优点。因此，本发明方法不仅可以与已有的基因激活方法相互补充，同时因为其独特的优势，更利于动物体内的应用，为crispr/cas9系统的基因治疗、肿瘤治疗等提供了新的应用前景，具有潜在的重要经济价值和意义。

附图说明

图1为prgen-cmv-vpr-linker-dcjcas9的质粒图谱。

图2为prgen-cmv-vpr-linker-minidcjcas9-hnhgsklinker的质粒图谱。

图3为prgen-cmv-vpr-linker-cjcas9wt的质粒图谱。

图4为prgen-cmv-vpr-s-linker-minidcjcas9-d2vp64new的质粒图谱，相比于prgen-cmv-vpr-linker-minidcjcas9-hnhgsklinker，该质粒的vp64进行了密码子优化，vpr整体缩小27个氨基酸序列，vpr与minidcjcas9之间的linker缩小33氨基酸。

图5为pu6-cj-sgrna的质粒图谱。

图6为pu6-cj-fsgrna的质粒图谱。

图7为qpcr检测结果图，显示融合蛋白vpr-dcjcas9可以在293t细胞中靶向激活hbg的表达。

图8为qpcr检测结果图，显示融合蛋白vpr-minidcjcas9可以在293t细胞中靶向激活hbg的表达。

图9为qpcr检测结果图，显示融合蛋白vpr-wtcjcas9可以在293t细胞中利用15bp导向rna靶向激活il1rn的表达。

图10为qpcr检测结果图，显示融合蛋白vpr-s-linker-minidcjcas9-d2vp64new可以在293t细胞中靶向激活hbg的表达。

图11为qpcr检测结果图，与wt向导rna相比，f骨架的向导rna可以提高cjcas9的靶向激活效果。

图12为qpcr检测结果图(a)和t7e1检测结果图(b)，显示融合蛋白vpr-wtcjcas9可以利用15bp导向rna靶向激活il1rn(a)，同时利用22bp导向rna靶向切割hbg(b)；ctr为仅转染融合蛋白vpr-wtcjcas9的载体，h22i15为同时转染融合蛋白vpr-wtcjcas9的载体和22bp的靶向hbg的向导rna以及15bp的靶向il1rn的向导rna。。

图13为为全转录本mrna测序散点图，结果显示融合蛋白vpr-dcjcas9靶向利用22bp导向rna靶向激活il1rn的表达，且具有高特异性(a)；融合蛋白vpr-wtdcjcas9靶向利用15bp导向rna靶向激活il1rn的表达，且具有高特异性(b)。i22为22bp的靶向il1rn的向导rna，i15为15bp的靶向il1rn的向导rna。

图14为cjcas9和vpr融合蛋白介导的基因激活系统的模式图(a)，以及不同亚型cjcas9和vpr融合蛋白的结构示意图(b)。

具体实施方式

为了使本发明的目的、技术方案和有益技术效果更加清晰，以下结合实施例，对本发明进行进一步详细说明。应当理解的是，本发明的实施方式不局限于以下的实施例介绍，实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。

实施例1

构建表达dcjcas9蛋白的载体：将购买所得的prgen-cmv-cjcas9(89752)质粒用nco1酶切得到3383bp片段为载体，同时以prgen-cmv-cjcas9为模板，进行pcr扩增，pcr1引物序列如下：

f1：gtattagtcatcgctattaccatggtgatgcggttttggc；(seqidno：11)

r1：agcgaaggccaggattctggccatgattcggatcccaagcttg；(seqidno：12)

pcr2引物序列如下：

f2：gccagaatcctggccttcgctatcggcatcagcagcatcg；(seqidno：13)

r2：accggctgtaggggtagatggcgtcgatttccagcatcttct；(seqidno：14)

pcr3引物序列如下：

f3：aagatgctggaaatcgacgccatctacccctacagccggt；(seqidno：15)

r3：agcaccttcagggcgaagtccattgtgtagatgggcacggcgta；(seqidno：16)

利用gibsonassembly试剂将上述4个片段进行连接得到prgen-cmv-dcjcas9质粒。

酶切体系50ul，具体包括：

载体质粒10ul，限制性内切酶酶1ul，catsmartbuffer(10x)5ul，ddh2o34ul。反应条件37℃水浴4h，并用0.8％琼脂糖凝胶电泳分析酶切产物，切胶回收相应的条带。

pcr体系50ul，具体包括：

反应体系：模板(浓度为1ng/ul)2.5ul，q5酶1ul，q5buffer(10x)5ul，q5enhancer5ul，引物f和r(浓度为10um)2.5ul，dntp4ul，ddh2o30ul。

反应条件：①98℃:2min，②98℃:30s，③58℃:30s，④72℃:30s，②③④循环40次，⑤72℃5min。

用0.8％琼脂糖凝胶电泳分析pcr产物，切胶回收条带。

gibsonassembly反应体系10μl，具体包括：

2xgibsonassemblymix3μl，pcr回收产物(所有pcr产物总和)2μl，载体酶切回收产物1μl，反应条件：50℃水浴1h。

将连接产物转化到top10大肠杆菌中，涂在含有100μg/ml氨苄青霉素的lb固体平板，37℃培养过夜，然后挑取单个克隆，37℃250rpm摇菌后提取质粒进行dna测序，由此筛选构建正确的载体质粒。

实施例2

构建表达vpr蛋白的载体：以pblu2ksp为载体，用kpn1和spe1酶切后得到的载体片段，以phrdsv40-scfv-gcn4-sfgfp-vp64-gb1-nls(60904)为模板，进行pcr扩增得到vp64，pcr1引物序列如下：

f1：ctcactatagggcgaattgggtaccgatgctttagacgattttga；(seqidno：17)

r1：cctcctcctccgcttcctcctagcatatctagatcaaagt；(seqidno：18)

以ms2-p65-hsf1_gfp(61423)为模板，进行pcr扩增得到p65，pcr2引物序列如下：

f2：actttgatctagatatgctaggaggaagcggaggaggagg；(seqidno：19)

r2：aggtcgctgccgctgccagacccactagaggaaatctgt；(seqidno：20)

以合成的rta序列为模板，进行pcr扩增得到rta，pcr3引物序列如下：

f3：acagatttcctctagtgggtctggcagcggcagcgacct；(seqidno：21)

r3：cggtggcggccgctctagaaaacagagatgtgtcgaaga；(seqidno：22)

利用gibsonassembly试剂将上述4个片段进行连接得到pblu-vpr质粒。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例3

构建表达融合蛋白vpr-dcjcas9的载体：以prgen-cmv-dcjcas9为载体，用bamh1酶切后得到的载体片段，以pblu-vpr为模板，进行pcr扩增得到vpr，pcr1引物序列如下：

f1：atagggagacccaagcttgggccaccatggatgctttagacgattttga；(seqidno：23)

r1：ggggagccgctgcccaggctaaacagagatgtgtcgaaga；(seqidno：24)

以pcag-dcas9-24xgcn4_v4-nls-p2a-bfp为模板，进行pcr扩增得到linker序列，pcr2引物序列如下：

f1：tcttcgacacatctctgtttagcctgggcagcggctccc；(seqidno：25)

r1：aggattctggccatgattcgtcctccagaacctccacctc；(seqidno：26)

利用gibsonassembly试剂将上述3个片段进行连接得到prgen-cmv-vpr-linker-dcjcas9质粒(如图1所示)。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例4

构建表达融合蛋白vpr-minidcjcas9的载体：以prgen-cmv-cjcas9(89752)为载体，用bamh1和hind111酶切后得到的载体片段，以prgen-cmv-vpr-linker-dcjcas9为模板，进行pcr扩增，pcr1引物序列如下：

f1：cagtccgtgggcgagtacctgtacaaagagtacttccaga；(seqidno：27)

r1：tcttggtgggcaggttcttggcccgctggctgtggtt；(seqidno：28)

pcr2引物序列如下：

f1：accacagccagcgggccaagaacctgcccaccaagaaa；(seqidno：29)

r1：atcaggatcagggagtccttgtacaggctaaagcagaac；(seqidno：30)

利用gibsonassembly试剂将上述3个片段进行连接得到prgen-cmv-vpr-linker-minidcjcas9-hnhgsklinker质粒(如图2所示)。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例5

构建表达融合蛋白vpr-wtcjcas9的载体：以prgen-cmv-vpr-linker-dcjcas9为载体，用bamh1和hind111酶切后得到的载体片段，以prgen-cmv-vpr-linker-dcjcas9为模板，进行pcr扩增得到vpr及linker片段，pcr引物序列如下：

f1：actcactatagggagacccaagcttgggccaccatggatg；(seqidno：31)

r1：aggattctggccatggttcgtcctccagaacctccacct；(seqidno：32)

利用gibsonassembly试剂将上述2个片段进行连接得到prgen-cmv-vpr-linker-wtcjcas9质粒(如图3所示)。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例6

构建表达融合蛋白vpr-s-linker-minidcjcas9-d2vp64new的载体：以prgen-cmv-vpr-linker-minidcjcas9-hnhgsklinker为载体，用bamh1和hind111酶切后得到的载体片段，以合成的密码子优化的vp64序列为模板，进行pcr扩增得到新的vp64序列，pcr引物序列如下：

f1：gggtttgccgccagaacacagaagcttgggccaccat；(seqidno：33)

r1：accatagtctgggccagcacggatcccaccttcctctttt；(seqidno：34)

以prgen-cmv-vpr-linker-dcjcas9为模板，进行pcr扩增得到缩小的p65、rta以及缩小的linker序列，pcr引物序列如下：

f1：aaaagaggaaggtgggatccgtgctggcccagactatggt；(seqidno：35)

r1：gatccacccggaccgttggatccaaacagagatgtgtcgaag；(seqidno：36)

利用gibsonassembly试剂将上述3个片段进行连接得到prgen-cmv-vpr-s-linker-minidcjcas9-d2vp64new质粒(如图4所示)。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例7

构建pu6-cj-fsgrna质粒，即包含f骨架序列的向导rna载体：以pu6-cj-sgrna(89753)(如图5所示)为载体，用bsmb1和spe1酶切后得到的载体片段，无模板，进行pcr扩增得到f骨架序列，pcr引物序列如下：

f1:aggatagaattcgatgtcgaaaaaaaagcggttttaggggattgtaaccccgcagagtcccgcaaactctttattt；(seqidno：37)

r1:gacgaaacaccgggagacgggatcccgtctccgtttaagtccctgaaaagggacttaaataaagagtttgcgggac；(seqidno：38)

利用gibsonassembly试剂将上述2个片段进行连接得到pu6-cj-fsgrna质粒(如图6所示)。

酶切体系、pcr扩增、gibsonassembly反应体系等具体步骤如实施例1。

实施例8

构建靶向基因hbg和il1rn的向导rna质粒：分别以pu6-cj-sgrna，pu6-cj-fsgrna为载体，用bsmb1酶切后得到的载体片段，用引物合成得到hbg和il1rn的靶向oliga序列：

hbg22bpoligo_f：aaacggcataggtccaggatttttga；(seqidno：39)

hbg22bpoligo_r：accgtcaaaaatcctggacctatgcc；(seqidno：40)

il1rn22bpoligo_f：aaacacatgcatgagctggcggcagt；(seqidno：41)

il1rn22bpoligo_r：accgactgccgccagctcatgcatgt；(seqidno：42)

il1rn15bpoligo_f：aaacacatgcatgagctgg；(seqidno：43)

il1rn15bpoligo_r：accgccagctcatgcatgt；(seqidno：44)

将对应的正反oliga序列退火后利用t4连接酶与对应的载体连接，即可得到靶向hbg和il1rn的向导rna质粒。

酶切体系等具体步骤如实施例1。

两条部分互补配对单链dna片段合成双链的dna片段(正反oliga序列退火)。具体步骤如下：

10ul100umoligo-f和10ul100umoligo-r预混于1.5mlep管中，用烧杯煮沸800ml的蒸馏水，将1.5mlep管置于沸水中5分钟，取出1.5mlep管室温放置过夜。

t4连接体系10μl，具体包括：

t4连接酶1μl，10xt4ligasebuffer1μl，oliga序列退火产物1μl，载体酶切回收产物2μl，ddh2o5μl，反应条件：25℃水浴1h。

将连接产物转化到top10大肠杆菌中，涂在含有100μg/ml氨苄青霉素的lb固体平板，37℃培养过夜，然后挑取单个克隆，37℃250rpm摇菌后提取质粒进行dna测序，由此筛选构建正确的载体质粒。

实施例9

转染细胞，将上述构建好的各种融合蛋白和相应的向导rna用转染试剂pei转进hek293t细胞，48小时后，收取细胞的rna进行qpcr检测，结果如图7-11所示。(ctr为仅转染相应的融合蛋白质粒，无向导rna)其中图7为融合蛋白vpr-dcjcas9和针对hbg的22bp靶向序列与wt骨架形成向导rna，结果显示融合蛋白vpr-dcjcas9可以在293t细胞中靶向激活hbg的表达；图8为融合蛋白vpr-minidcjcas9和针对hbg的22bp靶向序列与wt骨架形成向导rna，结果显示融合蛋白vpr-minidcjcas9可以在293t细胞中靶向激活hbg的表达；图9为融合蛋白vpr-wtcjcas9和针对il1rn靶向序列与wt骨架形成的15bp向导rna，结果显示融合蛋白vpr-wtcjcas9可以在293t细胞中利用15bp导向rna靶向激活il1rn的表达；图10为融合蛋白vpr-s-linker-minidcjcas9-d2vp64new和针对hbg的22bp靶向序列与wt骨架形成向导rna，结果显示融合蛋白vpr-s-linker-minidcjcas9-d2vp64new可以在293t细胞中靶向激活hbg的表达；图11为融合蛋白vpr-dcjcas9和针对hbg的22bp靶向序列与wt骨架或者f骨架形成向导rna，结果显示与wt向导rna相比，f骨架的向导rna可以提高cjcas9的靶向激活效果。

其中对于vpr-wtcjcas9融合蛋白系统，用转染试剂pei将表达融合蛋白vpr-wtcjcas9的载体和22bp的靶向hbg的向导rna以及15bp的靶向il1rn的向导rna同时转入hek293t细胞，48小时后，将其中一半的细胞收取细胞的rna进行qpcr检测，并将另一半的细胞用sds裂解法提取基因组dna，以基因组dna为模板进行pcr扩增，进行t7e1检测。结果如图12所示，vpr与wtcjcas9融合蛋白组成的激活系统可以同时实现基因的激活和切割。hbg位点pcr引物如下：

hbgf：tagcctttgccttgttccga；(seqidno：45)

hbgr：acacgcacatcttatgtcttagag。(seqidno：46)

本发明提供的各种vpr与cjcas9融合蛋白组成的激活系统均可在向导rna的引导下实现靶基因的高效激活。qpcr引物如下：

hbgqpcrf：gctgagtgaactgcactgtga；(seqidno：47)

hbgqpcrr：gaattctttgccgaaatgga；(seqidno：48)

il1rnqpcrf：ggaatccatggagggaagat；(seqidno：49)

il1rnqpcrr：tgttctcgctcaggtcagtg；(seqidno：50)

gapdhqpcrf：agaaggctggggctcatttg；(seqidno：51)

gapdhqpcrr：aggggccatccacagtcttc。(seqidno：52)

同时将收取的靶向il1rn基因的rna进行全转录组测序，结果如图13所示，结果显示融合蛋白vpr-dcjcas9靶向利用22bp导向rna靶向激活il1rn的表达，且具有高特异性(a)；融合蛋白vpr-wtdcjcas9靶向利用15bp导向rna靶向激活il1rn的表达，且具有高特异性(b)。本发明提供的vpr与cjcas9融合蛋白组成的激活系统具有强特异性。

虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

sequencelisting

<110>南方医科大学

<120>基于cjcas9和vpr核心结构域的融合蛋白、相应的dna靶向激活系统及其应用

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leulysvalpheglulystyrilevalseralaleuglygluval

122012251230

thrlysalaglupheargglnarggluaspphelyslyssergly

123512401245

proprolyslyslysarglysvaltyrprotyraspvalproasp

125012551260

tyrala

1265

<210>3

<211>1378

<212>prt

<213>人工序列

<400>3

aspalaleuaspasppheaspleuaspmetleuglyseraspalaleu

151015

aspasppheaspleuaspmetleuglyseraspalaleuaspaspphe

202530

aspleuaspmetleuglyseraspalaleuaspasppheaspleuasp

354045

metleuglyglyserglyglyglyglyserglyprolyslyslysarg

505560

lysvalalaalaalaglyserproserglyglnileserasnglnala

65707580

leualaleualaproserseralaprovalleualaglnthrmetval

859095

proserseralametvalproleualaglnproproalaproalapro

100105110

valleuthrproglyproproglnserleuseralaprovalprolys

115120125

serthrglnalaglygluglythrleuserglualaleuleuhisleu

130135140

glnpheaspalaaspgluaspleuglyalaleuleuglyasnserthr

145150155160

aspproglyvalphethraspleualaservalaspasnsergluphe

165170175

glnglnleuleuasnglnglyvalsermetserhisserthralaglu

180185190

prometleumetglutyrproglualailethrargleuvalthrgly

195200205

serglnargproproaspproalaprothrproleuglythrsergly

210215220

leuproasnglyleuserglyaspgluasppheserserilealaasp

225230235240

metasppheseralaleuleuserglnileserserserglysergly

245250255

serglyseraspleuserhisproproproargglyhisleuaspglu

260265270

leuthrthrthrleuglusermetthrgluaspleuasnleuaspser

275280285

proleuthrprogluleuasngluileleuaspthrpheleuasnasp

290295300

glucysleuleuhisalamethisileserthrglyleuserilephe

305310315320

aspthrserleupheserleuglyserglyserprolyslyslysarg

325330335

lysvalgluaspprolyslyslysarglysvalaspglyileglyser

340345350

glyserasnglyserserglyserasnglyproglyglyserglygly

355360365

glyglyserglyglyargthrmetalaargileleualapheaspile

370375380

glyileserserileglytrpalaphesergluasnaspgluleulys

385390395400

aspcysglyvalargilephethrlysvalgluasnprolysthrgly

405410415

gluserleualaleuproargargleualaargseralaarglysarg

420425430

leualaargarglysalaargleuasnhisleulyshisleuileala

435440445

asngluphelysleuasntyrgluasptyrglnserpheaspgluser

450455460

leualalysalatyrlysglyserleuileserprotyrgluleuarg

465470475480

pheargalaleuasngluleuleuserlysglnaspphealaargval

485490495

ileleuhisilealalysargargglytyraspaspilelysasnser

500505510

aspasplysglulysglyalaileleulysalailelysglnasnglu

515520525

glulysleualaasntyrglnservalglyglutyrleutyrlysglu

530535540

tyrpheglnlysphelysgluasnserlysgluphethrasnvalarg

545550555560

asnlyslysglusertyrgluargcysilealaglnserpheleulys

565570575

aspgluleulysleuilephelyslysglnargglupheglypheser

580585590

pheserlyslysphegluglugluvalleuservalalaphetyrlys

595600605

argalaleulysasppheserhisleuvalglyasncysserphephe

610615620

thraspglulysargalaprolysasnserproleualaphemetphe

625630635640

valalaleuthrargileileasnleuleuasnasnleulysasnthr

645650655

gluglyileleutyrthrlysaspaspleuasnalaleuleuasnglu

660665670

valleulysasnglythrleuthrtyrlysglnthrlyslysleuleu

675680685

glyleuseraspasptyrgluphelysglyglulysglythrtyrphe

690695700

ilegluphelyslystyrlysglupheilelysalaleuglygluhis

705710715720

asnleuserglnaspaspleuasngluilealalysaspilethrleu

725730735

ilelysaspgluilelysleulyslysalaleualalystyraspleu

740745750

asnglnasnglnileaspserleuserlysleugluphelysasphis

755760765

leuasnileserphelysalaleulysleuvalthrproleumetleu

770775780

gluglylyslystyraspglualacysasngluleuasnleulysval

785790795800

alaileasngluasplyslysasppheleuproalapheasngluthr

805810815

tyrtyrlysaspgluvalthrasnprovalvalleuargalailelys

820825830

glutyrarglysvalleuasnalaleuleulyslystyrglylysval

835840845

hislysileasnilegluleualaarggluvalglylysasnhisser

850855860

glnargalalysileglulysgluglnasngluasntyrlysalalys

865870875880

lysaspalagluleuglucysglulysleuglyleulysileasnser

885890895

lysasnileleulysleuargleuphelysgluglnlysgluphecys

900905910

alatyrserglyglulysilelysileseraspleuglnaspglulys

915920925

metleugluileasphisiletyrprotyrserargserpheaspasp

930935940

sertyrmetasnlysvalleuvalphethrlysglnasnglnglulys

945950955960

leuasnglnthrpropheglualapheglyasnaspseralalystrp

965970975

glnlysilegluvalleualalysasnleuprothrlyslysglnlys

980985990

argileleuasplysasntyrlysasplysgluglnlysasnphelys

99510001005

aspargasnleuasnaspthrargtyrilealaargleuvalleu

101010151020

asntyrthrlysasptyrleuasppheleuproleuseraspasp

102510301035

gluasnthrlysleuasnaspthrglnlysglyserlysvalhis

104010451050

valglualalysserglymetleuthrseralaleuarghisthr

105510601065

trpglypheseralalysaspargasnasnhisleuhishisala

107010751080

ileaspalavalileilealatyralaasnasnserilevallys

108510901095

alapheseraspphelyslysgluglngluserasnseralaglu

110011051110

leutyralalyslysilesergluleuasptyrlysasnlysarg

111511201125

lysphepheglupropheserglypheargglnlysvalleuasp

113011351140

lysileaspgluilephevalserlysprogluarglyslyspro

114511501155

serglyalaleuhisglugluthrphearglysgluglugluphe

116011651170

tyrglnsertyrglyglylysgluglyvalleulysalaleuglu

117511801185

leuglylysilearglysvalasnglylysilevallysasngly

119011951200

aspmetpheargvalaspilephelyshislyslysthrasnlys

120512101215

phetyralavalproiletyrthrmetaspphealaleulysval

122012251230

leuproasnlysalavalalaargserlyslysglygluilelys

123512401245

asptrpileleumetaspgluasntyrgluphecyspheserleu

125012551260

tyrlysaspserleuileleuileglnthrlysaspmetglnglu

126512701275

progluphevaltyrtyrasnalaphethrserserthrvalser

128012851290

leuilevalserlyshisaspasnlysphegluthrleuserlys

129513001305

asnglnlysileleuphelysasnalaasnglulysgluvalile

131013151320

alalysserileglyileglnasnleulysvalpheglulystyr

132513301335

ilevalseralaleuglygluvalthrlysalagluphearggln

134013451350

arggluaspphelyslysserglyproprolyslyslysarglys

135513601365

valtyrprotyraspvalproasptyrala

13701375

<210>4

<211>1205

<212>prt

<213>人工序列

<400>4

alaleuaspasppheaspleuaspmetleuglyseraspalaleuasp

151015

asppheaspleuaspmetleuglyseraspalaleuaspasppheasp

202530

leuaspmetleuglyseraspalaleuaspasppheaspleuaspmet

354045

leuglyglyserglyglyglyglyserglyprolyslyslysarglys

505560

valglyservalleualaglnthrmetvalproserseralametval

65707580

proleualaglnproproalaproalaprovalleuthrproglypro

859095

proglnserleuseralaprovalprolysserthrglnalaglyglu

100105110

glythrleuserglualaleuleuhisleuglnpheaspalaaspglu

115120125

aspleuglyalaleuleuglyasnserthraspproglyvalphethr

130135140

aspleualaservalaspasnserglupheglnglnleuleuasngln

145150155160

glyvalsermetserhisserthralagluprometleumetglutyr

165170175

proglualailethrargleuvalthrglyserglnargproproasp

180185190

proalaprothrproleuglythrserglyleuproasnglyleuser

195200205

glyaspgluasppheserserilealaaspmetasppheseralaleu

210215220

leuserglyserglyserglyseraspleuserhisproproproarg

225230235240

glyhisleuaspgluleuthrthrthrleuglusermetthrgluasp

245250255

leuasnleuaspserproleuthrprogluleuasngluileleuasp

260265270

thrpheleuasnaspglucysleuleuhisalamethisileserthr

275280285

glyleuserilepheaspthrserleupheglyserasnglyprogly

290295300

glyserglyglyglyglyserglyglyargilemetalaargileleu

305310315320

alaphealaileglyileserserileglytrpalaphesergluasn

325330335

aspgluleulysaspcysglyvalargilephethrlysvalgluasn

340345350

prolysthrglygluserleualaleuproargargleualaargser

355360365

alaarglysargleualaargarglysalaargleuasnhisleulys

370375380

hisleuilealaasngluphelysleuasntyrgluasptyrglnser

385390395400

pheaspgluserleualalysalatyrlysglyserleuileserpro

405410415

tyrgluleuargpheargalaleuasngluleuleuserlysglnasp

420425430

phealaargvalileleuhisilealalysargargglytyraspasp

435440445

ilelysasnseraspasplysglulysglyalaileleulysalaile

450455460

lysglnasngluglulysleualaasntyrglnservalglyglutyr

465470475480

leutyrlysglutyrpheglnlysphelysgluasnserlysgluphe

485490495

thrasnvalargasnlyslysglusertyrgluargcysilealagln

500505510

serpheleulysaspgluleulysleuilephelyslysglnargglu

515520525

pheglypheserpheserlyslysphegluglugluvalleuserval

530535540

alaphetyrlysargalaleulysasppheserhisleuvalglyasn

545550555560

cysserphephethraspglulysargalaprolysasnserproleu

565570575

alaphemetphevalalaleuthrargileileasnleuleuasnasn

580585590

leulysasnthrgluglyileleutyrthrlysaspaspleuasnala

595600605

leuleuasngluvalleulysasnglythrleuthrtyrlysglnthr

610615620

lyslysleuleuglyleuseraspasptyrgluphelysglyglulys

625630635640

glythrtyrpheilegluphelyslystyrlysglupheilelysala

645650655

leuglygluhisasnleuserglnaspaspleuasngluilealalys

660665670

aspilethrleuilelysaspgluilelysleulyslysalaleuala

675680685

lystyraspleuasnglnasnglnileaspserleuserlysleuglu

690695700

phelysasphisleuasnileserphelysalaleulysleuvalthr

705710715720

proleumetleugluglylyslystyraspglualacysasngluleu

725730735

asnleulysvalalaileasngluasplyslysasppheleuproala

740745750

pheasngluthrtyrtyrlysaspgluvalthrasnprovalvalleu

755760765

argalailelysglutyrarglysvalleuasnalaleuleulyslys

770775780

tyrglylysvalhislysileasnilegluleualaarggluvalgly

785790795800

lysasnhisserglnargalalysglyserlysasnleuprothrlys

805810815

lysglnlysargileleuasplysasntyrlysasplysgluglnlys

820825830

asnphelysaspargasnleuasnaspthrargtyrilealaargleu

835840845

valleuasntyrthrlysasptyrleuasppheleuproleuserasp

850855860

aspgluasnthrlysleuasnaspthrglnlysglyserlysvalhis

865870875880

valglualalysserglymetleuthrseralaleuarghisthrtrp

885890895

glypheseralalysaspargasnasnhisleuhishisalaileasp

900905910

alavalileilealatyralaasnasnserilevallysalapheser

915920925

aspphelyslysgluglngluserasnseralagluleutyralalys

930935940

lysilesergluleuasptyrlysasnlysarglysphepheglupro

945950955960

pheserglypheargglnlysvalleuasplysileaspgluilephe

965970975

valserlysprogluarglyslysproserglyalaleuhisgluglu

980985990

thrphearglysgluglugluphetyrglnsertyrglyglylysglu

99510001005

glyvalleulysalaleugluleuglylysilearglysvalasn

101010151020

glylysilevallysasnglyaspmetpheargvalaspilephe

102510301035

lyshislyslysthrasnlysphetyralavalproiletyrthr

104010451050

metaspphealaleulysvalleuproasnlysalavalalaarg

105510601065

serlyslysglygluilelysasptrpileleumetaspgluasn

107010751080

tyrgluphecyspheserleutyrlysaspserleuileleuile

108510901095

glnthrlysaspmetglngluprogluphevaltyrtyrasnala

110011051110

phethrserserthrvalserleuilevalserlyshisaspasn

111511201125

lysphegluthrleuserlysasnglnlysileleuphelysasn

113011351140

alaasnglulysgluvalilealalysserileglyileglnasn

114511501155

leulysvalpheglulystyrilevalseralaleuglygluval

116011651170

thrlysalaglupheargglnarggluaspphelyslyssergly

117511801185

proprolyslyslysarglysvaltyrprotyraspvalproasp

119011951200

tyrala

1205

<210>5

<211>4134

<212>dna

<213>人工序列

<400>5

gatgctttagacgattttgacttagatatgcttggttcagacgcgttagacgacttcgac60

ctagacatgttaggctcagatgcattggacgacttcgatttagatatgttgggctccgat120

gccctagatgactttgatctagatatgctaggaggaagcggaggaggaggtagcggacct180

aagaaaaagaggaaggtggcggccgctggatccccttcagggcagatcagcaaccaggcc240

ctggctctggcccctagctccgctccagtgctggcccagactatggtgccctctagtgct300

atggtgcctctggcccagccacctgctccagcccctgtgctgaccccaggaccaccccag360

tcactgagcgctccagtgcccaagtctacacaggccggcgaggggactctgagtgaagct420

ctgctgcacctgcagttcgacgctgatgaggacctgggagctctgctggggaacagcacc480

gatcccggagtgttcacagatctggcctccgtggacaactctgagtttcagcagctgctg540

aatcagggcgtgtccatgtctcatagtacagccgaaccaatgctgatggagtaccccgaa600

gccattacccggctggtgaccggcagccagcggccccccgaccccgctccaactcccctg660

ggaaccagcggcctgcctaatgggctgtccggagatgaagacttctcaagcatcgctgat720

atggactttagtgccctgctgtcacagatttcctctagtgggtctggcagcggcagcgac780

ctttcccatccgcccccaaggggccatctggatgagctgacaaccacacttgagtccatg840

accgaggatctgaacctggactcacccctgaccccggaattgaacgagattctggatacc900

ttcctgaacgacgagtgcctcttgcatgccatgcatatcagcacaggactgtccatcttc960

gacacatctctgtttagcctgggcagcggctcccccaagaaaaaacgcaaggtggaagat1020

cctaagaaaaagcggaaagtggacggcattggtagtgggagcaacggcagcagcggatcc1080

aacggtccgggtggatctggaggtggaggttctggaggacgaatcatggccagaatcctg1140

gccttcgctatcggcatcagcagcatcggctgggccttcagcgagaacgacgagctgaag1200

gactgcggcgtgcggatcttcaccaaggtggaaaaccccaagaccggcgagagcctggcc1260

ctgcccagaaggctggccagaagcgcccggaagagactggccagacggaaggcccggctg1320

aaccacctgaagcacctgatcgccaacgagttcaagctgaactacgaggactaccagagc1380

ttcgacgagtccctggccaaggcctacaagggcagcctgatcagcccctacgagctgcgg1440

ttccgggccctgaacgagctgctgagcaagcaggacttcgccagagtgatcctgcacatt1500

gccaagcggagaggctacgacgacatcaagaacagcgacgacaaagagaagggcgccatc1560

ctgaaggccatcaagcagaacgaggaaaagctggccaactaccagtccgtgggcgagtac1620

ctgtacaaagagtacttccagaagttcaaagagaacagcaaagaattcaccaacgtgcgg1680

aacaagaaagaaagctacgagcggtgtatcgcccagagcttcctgaaggatgagctgaag1740

ctgatcttcaagaagcagagagagttcggcttcagcttcagcaagaaattcgaggaagag1800

gtgctgagcgtcgccttctacaagagagccctgaaggacttcagccacctcgtgggcaac1860

tgcagcttcttcaccgacgagaagagagcccccaagaacagccccctggccttcatgttc1920

gtggccctgacccggatcatcaacctgctgaacaatctgaagaacaccgagggcatcctg1980

tacaccaaggacgacctgaacgccctgctgaatgaggtgctgaagaacggcaccctgacc2040

tacaagcagaccaagaagctgctgggcctgagcgacgactacgagtttaagggcgagaag2100

ggcacctacttcatcgagttcaagaagtacaaagagttcatcaaggccctgggcgagcac2160

aacctgagccaggacgatctgaatgagatcgccaaggacatcaccctgatcaaggacgag2220

attaagctgaagaaggccctggccaaatacgacctgaatcagaaccagatcgacagcctg2280

agcaagctggaattcaaggatcacctgaacatcagcttcaaggctctgaagctggtcacc2340

cccctgatgctggaaggcaagaagtacgacgaggcctgcaacgagctgaacctgaaggtg2400

gccatcaacgaggacaagaaggacttcctgcccgccttcaacgaaacctactacaaggac2460

gaagtgaccaaccccgtggtgctgcgggccatcaaagaataccggaaggtgctgaatgcc2520

ctgctcaagaaatacggcaaggtgcacaagatcaacatcgagctggcccgggaagtgggc2580

aagaaccacagccagcgggccaagatcgagaaagagcagaacgaaaactacaaggccaag2640

aaggacgctgagctggaatgcgagaagctgggactgaagatcaacagcaagaacatcctg2700

aagctgcggctgttcaaagaacagaaagagttctgcgcctacagcggcgagaagatcaag2760

atcagcgatctgcaggacgagaagatgctggaaatcgacgccatctacccctacagccgg2820

tccttcgacgacagctacatgaacaaggtgctggtgttcaccaaacagaaccaggaaaaa2880

ctgaaccagacccccttcgaggccttcggcaacgacagcgccaagtggcagaaaatcgag2940

gtgctggccaagaacctgcccaccaagaaacagaagagaatcctggacaagaattacaag3000

gacaaagagcagaagaacttcaaggaccggaacctgaacgacacccggtatatcgcccgg3060

ctggtgctgaactacacaaaggactacctggatttcctgcccctgtccgacgacgagaac3120

accaagctgaacgatacccagaaaggctccaaggtgcacgtggaagccaagagcggcatg3180

ctgaccagcgccctgagacacacctggggcttcagcgccaaggatcggaacaaccatctg3240

caccacgccatcgacgccgtgatcattgcctacgccaacaacagcatcgtgaaggccttc3300

tccgacttcaagaaagaacaggaaagcaacagcgccgagctgtacgccaagaagatctct3360

gagctggactacaagaacaagcggaagttcttcgagcccttcagcggcttccggcagaag3420

gtgctggataagatcgacgagatcttcgtgtccaagcccgagcggaagaagccctctggc3480

gccctgcacgaggaaaccttcagaaaagaggaagagttctaccagtcctacggcggcaaa3540

gaaggcgtgctgaaggccctcgagctgggcaagatcagaaaagtgaacggcaagatcgtg3600

aagaacggggacatgttccgggtggacatcttcaagcacaaaaagaccaacaagttctac3660

gccgtgcccatctacacaatggacttcgccctgaaggtgctgcccaacaaggccgtggcc3720

cggtccaagaagggcgagatcaaggactggattctgatggacgagaactacgagttctgc3780

tttagcctgtacaaggactccctgatcctgatccagaccaaggacatgcaggaacccgag3840

ttcgtctactacaacgccttcaccagcagcaccgtgtccctgatcgtgtctaagcacgac3900

aacaagttcgagacactgagcaagaaccagaagatcctgttcaagaacgccaacgagaaa3960

gaagtgatcgccaagagcatcggcatccagaatctgaaggtgttcgagaagtacatcgtg4020

tccgccctgggagaagtgacaaaggccgagttccggcagagagaggacttcaaaaagtcc4080

ggacctccaaagaaaaagagaaaagtatacccctacgacgtgcccgactacgcc4134

<210>6

<211>3795

<212>dna

<213>人工序列

<400>6

gatgctttagacgattttgacttagatatgcttggttcagacgcgttagacgacttcgac60

ctagacatgttaggctcagatgcattggacgacttcgatttagatatgttgggctccgat120

gccctagatgactttgatctagatatgctaggaggaagcggaggaggaggtagcggacct180

aagaaaaagaggaaggtggcggccgctggatccccttcagggcagatcagcaaccaggcc240

ctggctctggcccctagctccgctccagtgctggcccagactatggtgccctctagtgct300

atggtgcctctggcccagccacctgctccagcccctgtgctgaccccaggaccaccccag360

tcactgagcgctccagtgcccaagtctacacaggccggcgaggggactctgagtgaagct420

ctgctgcacctgcagttcgacgctgatgaggacctgggagctctgctggggaacagcacc480

gatcccggagtgttcacagatctggcctccgtggacaactctgagtttcagcagctgctg540

aatcagggcgtgtccatgtctcatagtacagccgaaccaatgctgatggagtaccccgaa600

gccattacccggctggtgaccggcagccagcggccccccgaccccgctccaactcccctg660

ggaaccagcggcctgcctaatgggctgtccggagatgaagacttctcaagcatcgctgat720

atggactttagtgccctgctgtcacagatttcctctagtgggtctggcagcggcagcgac780

ctttcccatccgcccccaaggggccatctggatgagctgacaaccacacttgagtccatg840

accgaggatctgaacctggactcacccctgaccccggaattgaacgagattctggatacc900

ttcctgaacgacgagtgcctcttgcatgccatgcatatcagcacaggactgtccatcttc960

gacacatctctgtttagcctgggcagcggctcccccaagaaaaaacgcaaggtggaagat1020

cctaagaaaaagcggaaagtggacggcattggtagtgggagcaacggcagcagcggatcc1080

aacggtccgggtggatctggaggtggaggttctggaggacgaatcatggccagaatcctg1140

gccttcgctatcggcatcagcagcatcggctgggccttcagcgagaacgacgagctgaag1200

gactgcggcgtgcggatcttcaccaaggtggaaaaccccaagaccggcgagagcctggcc1260

ctgcccagaaggctggccagaagcgcccggaagagactggccagacggaaggcccggctg1320

aaccacctgaagcacctgatcgccaacgagttcaagctgaactacgaggactaccagagc1380

ttcgacgagtccctggccaaggcctacaagggcagcctgatcagcccctacgagctgcgg1440

ttccgggccctgaacgagctgctgagcaagcaggacttcgccagagtgatcctgcacatt1500

gccaagcggagaggctacgacgacatcaagaacagcgacgacaaagagaagggcgccatc1560

ctgaaggccatcaagcagaacgaggaaaagctggccaactaccagtccgtgggcgagtac1620

ctgtacaaagagtacttccagaagttcaaagagaacagcaaagaattcaccaacgtgcgg1680

aacaagaaagaaagctacgagcggtgtatcgcccagagcttcctgaaggatgagctgaag1740

ctgatcttcaagaagcagagagagttcggcttcagcttcagcaagaaattcgaggaagag1800

gtgctgagcgtcgccttctacaagagagccctgaaggacttcagccacctcgtgggcaac1860

tgcagcttcttcaccgacgagaagagagcccccaagaacagccccctggccttcatgttc1920

gtggccctgacccggatcatcaacctgctgaacaatctgaagaacaccgagggcatcctg1980

tacaccaaggacgacctgaacgccctgctgaatgaggtgctgaagaacggcaccctgacc2040

tacaagcagaccaagaagctgctgggcctgagcgacgactacgagtttaagggcgagaag2100

ggcacctacttcatcgagttcaagaagtacaaagagttcatcaaggccctgggcgagcac2160

aacctgagccaggacgatctgaatgagatcgccaaggacatcaccctgatcaaggacgag2220

attaagctgaagaaggccctggccaaatacgacctgaatcagaaccagatcgacagcctg2280

agcaagctggaattcaaggatcacctgaacatcagcttcaaggctctgaagctggtcacc2340

cccctgatgctggaaggcaagaagtacgacgaggcctgcaacgagctgaacctgaaggtg2400

gccatcaacgaggacaagaaggacttcctgcccgccttcaacgaaacctactacaaggac2460

gaagtgaccaaccccgtggtgctgcgggccatcaaagaataccggaaggtgctgaatgcc2520

ctgctcaagaaatacggcaaggtgcacaagatcaacatcgagctggcccgggaagtgggc2580

aagaaccacagccagcgggccaagggaagcaagaacctgcccaccaagaaacagaagaga2640

atcctggacaagaattacaaggacaaagagcagaagaacttcaaggaccggaacctgaac2700

gacacccggtatatcgcccggctggtgctgaactacacaaaggactacctggatttcctg2760

cccctgtccgacgacgagaacaccaagctgaacgatacccagaaaggctccaaggtgcac2820

gtggaagccaagagcggcatgctgaccagcgccctgagacacacctggggcttcagcgcc2880

aaggatcggaacaaccatctgcaccacgccatcgacgccgtgatcattgcctacgccaac2940

aacagcatcgtgaaggccttctccgacttcaagaaagaacaggaaagcaacagcgccgag3000

ctgtacgccaagaagatctctgagctggactacaagaacaagcggaagttcttcgagccc3060

ttcagcggcttccggcagaaggtgctggataagatcgacgagatcttcgtgtccaagccc3120

gagcggaagaagccctctggcgccctgcacgaggaaaccttcagaaaagaggaagagttc3180

taccagtcctacggcggcaaagaaggcgtgctgaaggccctcgagctgggcaagatcaga3240

aaagtgaacggcaagatcgtgaagaacggggacatgttccgggtggacatcttcaagcac3300

aaaaagaccaacaagttctacgccgtgcccatctacaccatggacttcgccctgaaggtg3360

ctgcccaacaaggccgtggcccggtccaagaagggcgagatcaaggactggattctgatg3420

gacgagaactacgagttctgctttagcctgtacaaggactccctgatcctgatccagacc3480

aaggacatgcaggaacccgagttcgtctactacaacgccttcaccagcagcaccgtgtcc3540

ctgatcgtgtctaagcacgacaacaagttcgagacactgagcaagaaccagaagatcctg3600

ttcaagaacgccaacgagaaagaagtgatcgccaagagcatcggcatccagaatctgaag3660

gtgttcgagaagtacatcgtgtccgccctgggagaagtgacaaaggccgagttccggcag3720

agagaggacttcaaaaagtccggacctccaaagaaaaagagaaaagtatacccctacgac3780

gtgcccgactacgcc3795

<210>7

<211>4134

<212>dna

<213>人工序列

<400>7

gatgctttagacgattttgacttagatatgcttggttcagacgcgttagacgacttcgac60

ctagacatgttaggctcagatgcattggacgacttcgatttagatatgttgggctccgat120

gccctagatgactttgatctagatatgctaggaggaagcggaggaggaggtagcggacct180

aagaaaaagaggaaggtggcggccgctggatccccttcagggcagatcagcaaccaggcc240

ctggctctggcccctagctccgctccagtgctggcccagactatggtgccctctagtgct300

atggtgcctctggcccagccacctgctccagcccctgtgctgaccccaggaccaccccag360

tcactgagcgctccagtgcccaagtctacacaggccggcgaggggactctgagtgaagct420

ctgctgcacctgcagttcgacgctgatgaggacctgggagctctgctggggaacagcacc480

gatcccggagtgttcacagatctggcctccgtggacaactctgagtttcagcagctgctg540

aatcagggcgtgtccatgtctcatagtacagccgaaccaatgctgatggagtaccccgaa600

gccattacccggctggtgaccggcagccagcggccccccgaccccgctccaactcccctg660

ggaaccagcggcctgcctaatgggctgtccggagatgaagacttctcaagcatcgctgat720

atggactttagtgccctgctgtcacagatttcctctagtgggtctggcagcggcagcgac780

ctttcccatccgcccccaaggggccatctggatgagctgacaaccacacttgagtccatg840

accgaggatctgaacctggactcacccctgaccccggaattgaacgagattctggatacc900

ttcctgaacgacgagtgcctcttgcatgccatgcatatcagcacaggactgtccatcttc960

gacacatctctgtttagcctgggcagcggctcccccaagaaaaaacgcaaggtggaagat1020

cctaagaaaaagcggaaagtggacggcattggtagtgggagcaacggcagcagcggatcc1080

aacggtccgggtggatctggaggtggaggttctggaggacgaaccatggccagaatcctg1140

gccttcgacatcggcatcagcagcatcggctgggccttcagcgagaacgacgagctgaag1200

gactgcggcgtgcggatcttcaccaaggtggaaaaccccaagaccggcgagagcctggcc1260

ctgcccagaaggctggccagaagcgcccggaagagactggccagacggaaggcccggctg1320

aaccacctgaagcacctgatcgccaacgagttcaagctgaactacgaggactaccagagc1380

ttcgacgagtccctggccaaggcctacaagggcagcctgatcagcccctacgagctgcgg1440

ttccgggccctgaacgagctgctgagcaagcaggacttcgccagagtgatcctgcacatt1500

gccaagcggagaggctacgacgacatcaagaacagcgacgacaaagagaagggcgccatc1560

ctgaaggccatcaagcagaacgaggaaaagctggccaactaccagtccgtgggcgagtac1620

ctgtacaaagagtacttccagaagttcaaagagaacagcaaagaattcaccaacgtgcgg1680

aacaagaaagaaagctacgagcggtgtatcgcccagagcttcctgaaggatgagctgaag1740

ctgatcttcaagaagcagagagagttcggcttcagcttcagcaagaaattcgaggaagag1800

gtgctgagcgtcgccttctacaagagagccctgaaggacttcagccacctcgtgggcaac1860

tgcagcttcttcaccgacgagaagagagcccccaagaacagccccctggccttcatgttc1920

gtggccctgacccggatcatcaacctgctgaacaatctgaagaacaccgagggcatcctg1980

tacaccaaggacgacctgaacgccctgctgaatgaggtgctgaagaacggcaccctgacc2040

tacaagcagaccaagaagctgctgggcctgagcgacgactacgagtttaagggcgagaag2100

ggcacctacttcatcgagttcaagaagtacaaagagttcatcaaggccctgggcgagcac2160

aacctgagccaggacgatctgaatgagatcgccaaggacatcaccctgatcaaggacgag2220

attaagctgaagaaggccctggccaaatacgacctgaatcagaaccagatcgacagcctg2280

agcaagctggaattcaaggatcacctgaacatcagcttcaaggctctgaagctggtcacc2340

cccctgatgctggaaggcaagaagtacgacgaggcctgcaacgagctgaacctgaaggtg2400

gccatcaacgaggacaagaaggacttcctgcccgccttcaacgaaacctactacaaggac2460

gaagtgaccaaccccgtggtgctgcgggccatcaaagaataccggaaggtgctgaatgcc2520

ctgctcaagaaatacggcaaggtgcacaagatcaacatcgagctggcccgggaagtgggc2580

aagaaccacagccagcgggccaagatcgagaaagagcagaacgaaaactacaaggccaag2640

aaggacgctgagctggaatgcgagaagctgggactgaagatcaacagcaagaacatcctg2700

aagctgcggctgttcaaagaacagaaagagttctgcgcctacagcggcgagaagatcaag2760

atcagcgatctgcaggacgagaagatgctggaaatcgaccacatctacccctacagccgg2820

tccttcgacgacagctacatgaacaaggtgctggtgttcaccaaacagaaccaggaaaaa2880

ctgaaccagacccccttcgaggccttcggcaacgacagcgccaagtggcagaaaatcgag2940

gtgctggccaagaacctgcccaccaagaaacagaagagaatcctggacaagaattacaag3000

gacaaagagcagaagaacttcaaggaccggaacctgaacgacacccggtatatcgcccgg3060

ctggtgctgaactacacaaaggactacctggatttcctgcccctgtccgacgacgagaac3120

accaagctgaacgatacccagaaaggctccaaggtgcacgtggaagccaagagcggcatg3180

ctgaccagcgccctgagacacacctggggcttcagcgccaaggatcggaacaaccatctg3240

caccacgccatcgacgccgtgatcattgcctacgccaacaacagcatcgtgaaggccttc3300

tccgacttcaagaaagaacaggaaagcaacagcgccgagctgtacgccaagaagatctct3360

gagctggactacaagaacaagcggaagttcttcgagcccttcagcggcttccggcagaag3420

gtgctggataagatcgacgagatcttcgtgtccaagcccgagcggaagaagccctctggc3480

gccctgcacgaggaaaccttcagaaaagaggaagagttctaccagtcctacggcggcaaa3540

gaaggcgtgctgaaggccctcgagctgggcaagatcagaaaagtgaacggcaagatcgtg3600

aagaacggggacatgttccgggtggacatcttcaagcacaaaaagaccaacaagttctac3660

gccgtgcccatctacaccatggacttcgccctgaaggtgctgcccaacaaggccgtggcc3720

cggtccaagaagggcgagatcaaggactggattctgatggacgagaactacgagttctgc3780

tttagcctgtacaaggactccctgatcctgatccagaccaaggacatgcaggaacccgag3840

ttcgtctactacaacgccttcaccagcagcaccgtgtccctgatcgtgtctaagcacgac3900

aacaagttcgagacactgagcaagaaccagaagatcctgttcaagaacgccaacgagaaa3960

gaagtgatcgccaagagcatcggcatccagaatctgaaggtgttcgagaagtacatcgtg4020

tccgccctgggagaagtgacaaaggccgagttccggcagagagaggacttcaaaaagtcc4080

ggacctccaaagaaaaagagaaaagtatacccctacgacgtgcccgactacgcc4134

<210>8

<211>3615

<212>dna

<213>人工序列

<400>8

gcattggacgattttgatctggatatgctgggaagtgacgccctcgatgattttgacctt60

gacatgcttggtagtgatgcccttgatgactttgacctcgacatgctcggcagtgacgcc120

cttgatgatttcgacctggacatgctgggaggaagcggaggaggaggtagcggacctaag180

aaaaagaggaaggtgggatccgtgctggcccagactatggtgccctctagtgctatggtg240

cctctggcccagccacctgctccagcccctgtgctgaccccaggaccaccccagtcactg300

agcgctccagtgcccaagtctacacaggccggcgaggggactctgagtgaagctctgctg360

cacctgcagttcgacgctgatgaggacctgggagctctgctggggaacagcaccgatccc420

ggagtgttcacagatctggcctccgtggacaactctgagtttcagcagctgctgaatcag480

ggcgtgtccatgtctcatagtacagccgaaccaatgctgatggagtaccccgaagccatt540

acccggctggtgaccggcagccagcggccccccgaccccgctccaactcccctgggaacc600

agcggcctgcctaatgggctgtccggagatgaagacttctcaagcatcgctgatatggac660

tttagtgccctgctgagtgggtctggcagcggcagcgacctttcccatccgcccccaagg720

ggccatctggatgagctgacaaccacacttgagtccatgaccgaggatctgaacctggac780

tcacccctgaccccggaattgaacgagattctggataccttcctgaacgacgagtgcctc840

ttgcatgccatgcatatcagcacaggactgtccatcttcgacacatctctgtttggatcc900

aacggtccgggtggatctggaggtggaggttctggaggacgaatcatggccagaatcctg960

gccttcgctatcggcatcagcagcatcggctgggccttcagcgagaacgacgagctgaag1020

gactgcggcgtgcggatcttcaccaaggtggaaaaccccaagaccggcgagagcctggcc1080

ctgcccagaaggctggccagaagcgcccggaagagactggccagacggaaggcccggctg1140

aaccacctgaagcacctgatcgccaacgagttcaagctgaactacgaggactaccagagc1200

ttcgacgagtccctggccaaggcctacaagggcagcctgatcagcccctacgagctgcgg1260

ttccgggccctgaacgagctgctgagcaagcaggacttcgccagagtgatcctgcacatt1320

gccaagcggagaggctacgacgacatcaagaacagcgacgacaaagagaagggcgccatc1380

ctgaaggccatcaagcagaacgaggaaaagctggccaactaccagtccgtgggcgagtac1440

ctgtacaaagagtacttccagaagttcaaagagaacagcaaagaattcaccaacgtgcgg1500

aacaagaaagaaagctacgagcggtgtatcgcccagagcttcctgaaggatgagctgaag1560

ctgatcttcaagaagcagagagagttcggcttcagcttcagcaagaaattcgaggaagag1620

gtgctgagcgtcgccttctacaagagagccctgaaggacttcagccacctcgtgggcaac1680

tgcagcttcttcaccgacgagaagagagcccccaagaacagccccctggccttcatgttc1740

gtggccctgacccggatcatcaacctgctgaacaatctgaagaacaccgagggcatcctg1800

tacaccaaggacgacctgaacgccctgctgaatgaggtgctgaagaacggcaccctgacc1860

tacaagcagaccaagaagctgctgggcctgagcgacgactacgagtttaagggcgagaag1920

ggcacctacttcatcgagttcaagaagtacaaagagttcatcaaggccctgggcgagcac1980

aacctgagccaggacgatctgaatgagatcgccaaggacatcaccctgatcaaggacgag2040

attaagctgaagaaggccctggccaaatacgacctgaatcagaaccagatcgacagcctg2100

agcaagctggaattcaaggatcacctgaacatcagcttcaaggctctgaagctggtcacc2160

cccctgatgctggaaggcaagaagtacgacgaggcctgcaacgagctgaacctgaaggtg2220

gccatcaacgaggacaagaaggacttcctgcccgccttcaacgaaacctactacaaggac2280

gaagtgaccaaccccgtggtgctgcgggccatcaaagaataccggaaggtgctgaatgcc2340

ctgctcaagaaatacggcaaggtgcacaagatcaacatcgagctggcccgggaagtgggc2400

aagaaccacagccagcgggccaagggaagcaagaacctgcccaccaagaaacagaagaga2460

atcctggacaagaattacaaggacaaagagcagaagaacttcaaggaccggaacctgaac2520

gacacccggtatatcgcccggctggtgctgaactacacaaaggactacctggatttcctg2580

cccctgtccgacgacgagaacaccaagctgaacgatacccagaaaggctccaaggtgcac2640

gtggaagccaagagcggcatgctgaccagcgccctgagacacacctggggcttcagcgcc2700

aaggatcggaacaaccatctgcaccacgccatcgacgccgtgatcattgcctacgccaac2760

aacagcatcgtgaaggccttctccgacttcaagaaagaacaggaaagcaacagcgccgag2820

ctgtacgccaagaagatctctgagctggactacaagaacaagcggaagttcttcgagccc2880

ttcagcggcttccggcagaaggtgctggataagatcgacgagatcttcgtgtccaagccc2940

gagcggaagaagccctctggcgccctgcacgaggaaaccttcagaaaagaggaagagttc3000

taccagtcctacggcggcaaagaaggcgtgctgaaggccctcgagctgggcaagatcaga3060

aaagtgaacggcaagatcgtgaagaacggggacatgttccgggtggacatcttcaagcac3120

aaaaagaccaacaagttctacgccgtgcccatctacacaatggacttcgccctgaaggtg3180

ctgcccaacaaggccgtggcccggtccaagaagggcgagatcaaggactggattctgatg3240

gacgagaactacgagttctgctttagcctgtacaaggactccctgatcctgatccagacc3300

aaggacatgcaggaacccgagttcgtctactacaacgccttcaccagcagcaccgtgtcc3360

ctgatcgtgtctaagcacgacaacaagttcgagacactgagcaagaaccagaagatcctg3420

ttcaagaacgccaacgagaaagaagtgatcgccaagagcatcggcatccagaatctgaag3480

gtgttcgagaagtacatcgtgtccgccctgggagaagtgacaaaggccgagttccggcag3540

agagaggacttcaaaaagtccggacctccaaagaaaaagagaaaagtatacccctacgac3600

gtgcccgactacgcc3615

<210>9

<211>80

<212>dna

<213>人工序列

<400>9

gttttagtccctgaaaagggactaaaataaagagtttgcgggactctgcggggttacaat60

cccctaaaaccgcttttttt80

<210>10

<211>80

<212>dna

<213>人工序列

<400>10

gtttaagtccctgaaaagggacttaaataaagagtttgcgggactctgcggggttacaat60

cccctaaaaccgcttttttt80

<210>11

<211>40

<212>dna

<213>人工序列

<400>11

gtattagtcatcgctattaccatggtgatgcggttttggc40

<210>12

<211>43

<212>dna

<213>人工序列

<400>12

agcgaaggccaggattctggccatgattcggatcccaagcttg43

<210>13

<211>40

<212>dna

<213>人工序列

<400>13

gccagaatcctggccttcgctatcggcatcagcagcatcg40

<210>14

<211>42

<212>dna

<213>人工序列

<400>14

accggctgtaggggtagatggcgtcgatttccagcatcttct42

<210>15

<211>40

<212>dna

<213>人工序列

<400>15

aagatgctggaaatcgacgccatctacccctacagccggt40

<210>16

<211>44

<212>dna

<213>人工序列

<400>16

agcaccttcagggcgaagtccattgtgtagatgggcacggcgta44

<210>17

<211>45

<212>dna

<213>人工序列

<400>17

ctcactatagggcgaattgggtaccgatgctttagacgattttga45

<210>18

<211>40

<212>dna

<213>人工序列

<400>18

cctcctcctccgcttcctcctagcatatctagatcaaagt40

<210>19

<211>40

<212>dna

<213>人工序列

<400>19

actttgatctagatatgctaggaggaagcggaggaggagg40

<210>20

<211>39

<212>dna

<213>人工序列

<400>20

aggtcgctgccgctgccagacccactagaggaaatctgt39

<210>21

<211>39

<212>dna

<213>人工序列

<400>21

acagatttcctctagtgggtctggcagcggcagcgacct39

<210>22

<211>39

<212>dna

<213>人工序列

<400>22

cggtggcggccgctctagaaaacagagatgtgtcgaaga39

<210>23

<211>49

<212>dna

<213>人工序列

<400>23

atagggagacccaagcttgggccaccatggatgctttagacgattttga49

<210>24

<211>40

<212>dna

<213>人工序列

<400>24

ggggagccgctgcccaggctaaacagagatgtgtcgaaga40

<210>25

<211>39

<212>dna

<213>人工序列

<400>25

tcttcgacacatctctgtttagcctgggcagcggctccc39

<210>26

<211>40

<212>dna

<213>人工序列

<400>26

aggattctggccatgattcgtcctccagaacctccacctc40

<210>27

<211>40

<212>dna

<213>人工序列

<400>27

cagtccgtgggcgagtacctgtacaaagagtacttccaga40

<210>28

<211>37

<212>dna

<213>人工序列

<400>28

tcttggtgggcaggttcttggcccgctggctgtggtt37

<210>29

<211>38

<212>dna

<213>人工序列

<400>29

accacagccagcgggccaagaacctgcccaccaagaaa38

<210>30

<211>39

<212>dna

<213>人工序列

<400>30

atcaggatcagggagtccttgtacaggctaaagcagaac39

<210>31

<211>40

<212>dna

<213>人工序列

<400>31

actcactatagggagacccaagcttgggccaccatggatg40

<210>32

<211>39

<212>dna

<213>人工序列

<400>32

aggattctggccatggttcgtcctccagaacctccacct39

<210>33

<211>37

<212>dna

<213>人工序列

<400>33

gggtttgccgccagaacacagaagcttgggccaccat37

<210>34

<211>40

<212>dna

<213>人工序列

<400>34

accatagtctgggccagcacggatcccaccttcctctttt40

<210>35

<211>40

<212>dna

<213>人工序列

<400>35

aaaagaggaaggtgggatccgtgctggcccagactatggt40

<210>36

<211>42

<212>dna

<213>人工序列

<400>36

gatccacccggaccgttggatccaaacagagatgtgtcgaag42

<210>37

<211>76

<212>dna

<213>人工序列

<400>37

aggatagaattcgatgtcgaaaaaaaagcggttttaggggattgtaaccccgcagagtcc60

cgcaaactctttattt76

<210>38

<211>76

<212>dna

<213>人工序列

<400>38

gacgaaacaccgggagacgggatcccgtctccgtttaagtccctgaaaagggacttaaat60

aaagagtttgcgggac76

<210>39

<211>26

<212>dna

<213>人工序列

<400>39

aaacggcataggtccaggatttttga26

<210>40

<211>26

<212>dna

<213>人工序列

<400>40

accgtcaaaaatcctggacctatgcc26

<210>41

<211>26

<212>dna

<213>人工序列

<400>41

aaacacatgcatgagctggcggcagt26

<210>42

<211>26

<212>dna

<213>人工序列

<400>42

accgactgccgccagctcatgcatgt26

<210>43

<211>19

<212>dna

<213>人工序列

<400>43

aaacacatgcatgagctgg19

<210>44

<211>19

<212>dna

<213>人工序列

<400>44

accgccagctcatgcatgt19

<210>45

<211>20

<212>dna

<213>人工序列

<400>45

tagcctttgccttgttccga20

<210>46

<211>24

<212>dna

<213>人工序列

<400>46

acacgcacatcttatgtcttagag24

<210>47

<211>21

<212>dna

<213>人工序列

<400>47

gctgagtgaactgcactgtga21

<210>48

<211>20

<212>dna

<213>人工序列

<400>48

gaattctttgccgaaatgga20

<210>49

<211>20

<212>dna

<213>人工序列

<400>49

ggaatccatggagggaagat20

<210>50

<211>20

<212>dna

<213>人工序列

<400>50

tgttctcgctcaggtcagtg20

<210>51

<211>20

<212>dna

<213>人工序列

<400>51

agaaggctggggctcatttg20

<210>52

<211>20

<212>dna

<213>人工序列

<400>52

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