CN106957859A - 一种用于拯救麻疹病毒、重组麻疹病毒的系统以及方法 - Google Patents
一种用于拯救麻疹病毒、重组麻疹病毒的系统以及方法 Download PDFInfo
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Abstract
本发明公开了一种用于拯救麻疹病毒及表达GFP的重组麻疹病毒的系统、方法及用途，本发明的系统包括如下成分：1)含有麻疹病毒反基因组全长的转录质粒pT7MVS191或pT7MVS191‑ATU‑GFP；2)辅助质粒1，其含有麻疹病毒的核衣壳蛋白的编码基因；3)辅助质粒2，其含有麻疹病毒的磷蛋白的编码基因；4)辅助质粒3，其含有麻疹病毒的RNA聚合酶的编码基因。本发明还提供了一种拯救麻疹病毒及重组麻疹病毒的方法。本发明的系统和方法可以高效拯救麻疹病毒及重组麻疹病毒，可以用于治疗性或预防性重组麻疹病毒产品的开发及应用。
Description
技术领域
本发明属于一种用于拯救麻疹病毒、重组麻疹病毒的系统以及方法
背景技术
麻疹病毒(measles virus)是麻疹的病原体，分类上属于副粘病毒科麻疹病毒属。麻疹是儿童常见的一种急性传染病，其传染性很强，以皮丘疹、发热及呼吸道症状为特征。若无并发症，愈后良好。我国自60年代初应用减毒活疫苗以来，儿童的发病率显著下降。但在发展中国家仍是儿童死亡的一个主要原因。在天花灭绝后，WHO已将麻疹列为计划消灭的传染病之一。另外发现亚急性硬化性全脑炎(subacute sclerosing panencephalitits，SSPE)与麻疹病毒有关。
MV-S191是我国麻疹疫苗的生产株之一，已有研究表明麻疹接种后3～10年以上儿童抗体阳性率高,阳性率80.6％，抗体水平均值为108.42IU/L。提示接种麻疹疫苗后远期较高的保护性抗体水平，说明MV-S191麻疹疫苗具有良好的免疫原性。MV-S191在鸡胚成纤维细胞中连续传代35代，全基因组序列未发生任何核苷酸和氨基酸改变，未发现由回复突变产生的致病性毒株；MV感染宿主细胞，在宿主细胞细胞质中完成复制，排除整合到宿主染色体上的可能性；以上两点充分体现了MV疫苗良好的遗传稳定性和安全性。但是，目前麻疹疫苗的制备工艺复杂，需要在不同的细胞系中传代许多次才能得到减毒的毒株，费时费力。
公开号为103160473的专利申请公开了一种用于拯救麻疹病毒的方法，但是该方法利用宿主细胞RNA聚合酶II，在293-T细胞中通过CMV启动子引导病毒基因组转录进行11组病毒拯救实验，仅组5，组7和组8成功拯救出了麻疹病毒，难以稳定获得。
发明内容
本发明的目的在于提供一种新的用于拯救麻疹病毒的方法。
本发明首先提供了一种用于拯救麻疹病毒或重组麻疹病毒的系统，它包括如下成分：
1)含有麻疹病毒反基因组全长的转录质粒；
2)辅助质粒1，其含有麻疹病毒的核衣壳蛋白的编码基因；
3)辅助质粒2，其含有麻疹病毒的磷蛋白的编码基因；
4)辅助质粒3，其含有麻疹病毒的RNA聚合酶的编码基因。
其中，所述麻疹病毒为减毒麻疹疫苗株沪191；所述的重组麻疹病毒为表达GFP的重组麻疹病毒沪191。
其中，所述含有麻疹病毒反基因组全长的转录质粒为含有麻疹病毒反基因组全长的质粒pT7MVS191或pT7MVS191-ATU1-GFP；其中，所述pT7MVS191的核苷酸序列如SEQ ID NO.1所示；所述pT7MVS191-ATU1-GFP的核苷酸序列如SEQ ID NO.2所示。
其中，所述辅助质粒1的核苷酸序列如SEQ ID NO.3所示。
其中，所述辅助质粒2的核苷酸序列如SEQ ID NO.4所示。
其中，所述辅助质粒3的核苷酸序列如SEQ ID NO.5所示。
本发明还提供了前述拯救麻疹病毒及重组麻疹病毒的方法，它是采用前述的系统进行拯救，步骤如下：
(1)将前述系统中的质粒共转染细胞汇合度为60％～90％时的辅助细胞；
(2)37℃培养，热休克，37℃继续培养3d，得培养的上清混合物。
(3)取步骤(2)的上清混合物，感染宿主细胞，回收表达的病毒颗粒。
步骤(1)中，所述辅助细胞为表达T7RNA聚合酶的BSR T7细胞。
步骤(2)中，所述热休克的温度为42-44℃，优选温度为43.5℃；所述热休克时间为1-4小时，优选3小时。
步骤(3)中，所述的宿主细胞为Veroslam细胞、人二倍体细胞或者鸡胚成纤维细胞。
本发明还提供了前述的方法在制备适用于治疗性和/或预防性的感染性麻疹病毒和/或重组麻疹病毒中的用途。
MV基因组由连续独立的转录单元---核衣壳蛋白(N)、磷酸化蛋白(P/C/V)、基质蛋白(M)、融膜蛋白(F)、血凝素蛋白(H)及L蛋白组成，这些基因之间存在一段基因间隔区域，该区的核苷酸变化不影响病毒的转录和复制。已有多项研究在P和M基因以及H和L之间的基因间隔区域引入转录单元表达外源基因，并已经应用到疫苗开发中。此外，由于MV核衣壳呈螺旋对称，可携带的外源基因达MV基因组大小的30％，经12轮传代后，96％的病毒感染细胞可稳定表达外源基因。MV感染免疫系统的巨噬细胞和树突状细胞等抗原递呈细胞后，外源基因可通过相应免疫应答途径产生抗体。
本发明建立了可以拯救麻疹病毒沪191毒株、表达GFP的重组麻疹病毒的系统，该系统进行12组实验，均得到了具有感染力的麻疹病毒和重组麻疹病毒，拯救效率高，稳定性好，应用前景非常优良。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施实例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1麻疹病毒沪-191株分段示意
图2载体pT7-IRES His-C DNA的改造示意图
图3pT7MVS191的构建示意图
图4pT7MVS191-ATU-GFP的构建示意图
图5pT7-MV-N的构建示意图
图6pT7-MV-P的构建示意图
图7pT7-MV-L的构建示意图
图8改造后载体pT7-MCS的鉴定结果，1：Asc I、Ssp I双酶切载体pT7-MCS；2：DL15000DNA分子量标准
图9pT7MVS191、pT7MVS191-ATU-GFP的鉴定结果
1-3：pT7MVS191-ATU-GFP的限制性酶切分析：1：SpeI、2：NcoI、3：EcoRI、4：GFP的PCR产物5：DL5000DNA分子量标准、6：DL15000DNA分子量标准、7-8：pT7MVS191的限制性酶切分析7：SpeI、8：EcoRI、9：NcoI
图10MVS191拯救实验，A:光学显微镜B:荧光显微镜C:pT7MVS191示意图
图11MVS191-ATU-GFP拯救实验，A:光学显微镜B:荧光显微镜C:pT7MVS191-ATU-GFP示意图
图12RT-PCR鉴定病毒感染，1-3:第1、2、3代毒种感染的Veroslam细胞的MVF1PCR鉴定结果；4-6:第1、2、3代毒种感染的Veroslam细胞的MVF4PCR鉴定结果；7：DL5000DNA分子量标准
图13本发明拯救麻疹病毒或表达GFP的重组麻疹病毒的系统
具体实施方式
实施例1本发明拯救麻疹病毒或表达GFP的重组麻疹病毒载体的构建
1材料与方法
1.1载体、毒株来源
麻疹病毒沪191来源于上海生物制品研究所。pT7‐IRES His‐C DNA购自Takara公司；载体pGEM-T购自美国Promega公司。
1.2主要试剂
高纯度病毒RNA提取试剂盒购自Roche公司；phusion高保真DNA聚合酶、T4DNA连接酶购自NEB公司；随机引物、Superscript III反转录酶、细胞转染试剂LipofectamineTM2000购自美国Invitrogen公司；胶回收试剂盒购自promega公司；质粒提取试剂盒购自omega公司；琼脂糖、DNA marker购自Takara公司；其他试剂均为国产试剂。
1.3病毒RNA提取和RT-PCR
取冻干疫苗株，水溶解后，按照Roche高纯度病毒RNA提取试剂盒说明书所示方法提取麻疹病毒沪-191株的基因组RNA，以随机引物将基因组RNA逆转录为cDNA。反应条件为：随机引物1ul，RNA11ul，dNTPs 1ul混合后，65℃反应5min，冰浴1min。然后加入5×FSbuffer 4ul，0.1M DTT 1ul，RNaseOUT 1ul，Superscript III 1ul，55℃反应60min；再70℃反应15min。以此cDNA为模板，用Phusion高保真DNA聚合酶将病毒反基因组分为7个片段进行聚合酶链反应(PCR)扩增。7个片段对应的引物及所含酶切位点如表1所示。
麻疹病毒沪-191株分段示意图如图1所示。
表1.麻疹病毒沪-191株分段克隆引物
1.4载体pT7-IRES His-C DNA的改造
如图2所示，以pT7-IRES His-C DNA载体为模板，以pT7-IRES-NotI：ATCCGCGGCCGCTAATTCAC以及pT7-IRES-NheI：
TAGCTAGCAAGCTTGGCGTAATCATG为上下游引物，PCR扩增去除pT7-IRES His C DNA载体EMCV-IRES序列及SacII酶切位点。合成NheI-AscI-SacII-XmaI-PacI-EcoRI-KpnI-BsrGI-NotI限制性内切酶的多克隆位点序列(MCS)：
GCTAGCTAGTTGGCGCGCCAATCCCCGCGGGGATCCCCCCGGGGGGACCTTAATTAAGGCCGGAATTCCGGCGGGGTACCCCGGCGCTGTACAGCGCATAAGAATGCGGCCGC。将多克隆位点序列(MCS)通过NheI和NotI酶切位点与载体PCR扩增产物连接，经转化后，挑取菌落进行扩大培养，提取质粒，选取合适的限制性内切酶对质粒进行鉴定。改造后的载体命名为pT7-MCS。
用替换后多克隆位点(MCS)上的内切酶Asc I和载体上的单一酶切位点Ssp I对改造后的pT7-MCS进行双酶切鉴定，酶切后的长度为691bp和2300bp酶切鉴定结果见图7。
1.5病毒反基因组PCR产物的克隆
扩增的7段PCR产物经1％琼脂糖凝胶电泳后，胶回收目的条带，回收产物与载体pGEM-T进行连接。转化JM109感受态，挑取菌落进行扩大培养，小提质粒。测序确认插入片段与麻疹病毒沪-191株标准序列(Genbank access No：FJ416067.1)完全相同后，将质粒分别命名为pT-MVF1、pT-MVF2、pT-MVF3、pT-MVF4、pT-MVF5、pT-MVF6以及pT-MVF7。
根据Kanazaw a等[7]所述的锤头核酸酶序列(HamRz)的结构和作用原理在载体pT-MVF1的5'端插入T7启动子以及HamRz。用AscI和BamHI酶切合成序列：
5’-GGCGCGCCTAATACGACTCACTATAGGGCCAACTTTGTTTGGTCTGATGAGTCCGTGAGGACGAAACCCGGGTCACCAAACAAAGTTGGGTAAGGATAGTTCAATCAATGATCATCTTCTAGTGCACTTAGGATTCAAGATCCTATTATCAGGGACAAGAGCAGGATTAGGGATATCCGAGATGGCCACACTTTTAAGGAGCTTAGCATTGTTCAAAAGAAACAAGGACAAACCACCCATTACATCAGGATCC-3’与经AscI和BamHI酶切的pT-MVF1在T4DNA连接酶作用下连接，转化JM109感受态。得到的质粒命名为pT7-MVF1-HamRz；
根据Been等[8]所述的丁型肝炎病毒核酶序列(HdvRz)的结构和作用原理在载体pT-MVF7 3'端插入T7终止子以及HdvRz。用BspEI和NotI酶切合成序列：
5’-TCCGGAACCCTAATCCTGCCCTAGGTGGTTAGGCATTATTTGCAATATATTAAAGAAAACTTTGAAAATACGAAGTTTCTATTCCCAGCTTTGTCTGGTGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGGGACGCGGCCGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGCCGC-3’与经BspEI和NotI酶切的pT-MVF7在T4DNA连接酶作用下连接，转化JM109感受态。得到的质粒命名为pT-MVF7-HdvRz。
1.6构建病毒全长cDNA的转录质粒pT7MVS191
pT7MVS191的构建示意图如图3所示。
将片段MVF2与pT7-MCS连接，并依次连接片段MVF3得到质粒pT7-MVF23，而后将片段MVF1(HamRz)克隆至pT7-MVF23，得到麻疹病毒反基因组5’半分子质粒，命名为pT7MVS191-5'half；将片段MVF4、MVF5、MVF6、MVF7-HdvRz先后克隆至pT7-MCS，得到麻疹病毒反基因组3’半分子质粒，命名为pT7MVS191-3'half。将3’半分子用PacI和NotI酶切后克隆至5’半分子质粒，即得到含有麻疹病毒反基因组全长的质粒，命名为pT7MVS191(图3)。
1.7构建表达GFP的病毒全长cDNA的转录质粒pT7MVS191-ATU-GFP
pT7MVS191-ATU-GFP的构建示意图如图4所示。
突变掉pT7-MCS-MVF2载体上的AscI酶切位点。用SalI和SpeI酶切人工合成的附加转录单位(ATU)序列：
5’-gtcgacccaactagcCTACCCTCCATCATTGTTATAAAAAACTTAGGAACCAGGTCCACACAGCCGCCAGCCCATCAACGCGTACGATGGTGAGCAAGGGCGAGGAGCTGTTCACCACTCTCGGCATGGACGAGCTGTACAAGTAGGCGCGCAGCGCTTAGACGTCTCGCGACTAGT-3’克隆至载体pT7-MVF2，将此质粒命名为pT7-MVF2-ATU。将MVF3片段与pT7-MVF2-ATU载体连接，构建pT7-MVF23-ATU。该质粒用BsiWI和BssHII双酶切后与GFP连接，构建pTMVS191-5'half-ATU-GFP。然后按前述方式构建含有ATU的麻疹病毒反基因组5’半分子，命名为pT7MVS191-5'half-ATU-GFP。将前述3’半分子pTMVS191-3'half用PacI和NotI酶切后克隆至5’半分子质粒pT7MVS191-5'half-ATU-GFP，即得到含有GFP的麻疹病毒反基因组全长序列的质粒，命名为pT7MVS191-ATU-GFP。
酶切鉴定pT7MVS191、pT7MVS191-ATU-GFP的结果：
DNAman软件对pT7MVS191载体序列进行单酶切分析，SpeI可将该质粒酶切成2个片段，片段大小为：5802bp，13028bp。NcoI可将该质粒酶切成4个片段，片段大小为：936bp，1898bp，2681bp，13315bp。EcoRI可将该质粒酶切成3个片段，片段大小为：411bp，5189bp，13230bp。电泳结果如图9。酶切结果与理论序列酶切分析结果一致。
DNAman软件对pT7MVS191-ATU-GFP载体序列进行酶切鉴定，SpeI可将该质粒酶切成2个片段，片段大小为：5802bp，13892bp。NcoI可将该质粒酶切成4个片段，片段大小为：936bp，1898bp，2681bp，14179bp。EcoRI可将该质粒酶切成3个片段，片段大小为：411bp，660bp，5189bp，13230bp。用特异性上下游引物扩增GFP片段，如图9(Lane 4)可见760bp的目的条带。将酶切鉴定正确的全长基因送样测序，pT7MVS191-ATU-GFP质粒中的GFP编码区序列,经与GenBank上发布的GFP(登录号:U17997)中的相应核苷酸序列进行比对后确认所测的序列和GenBank上发布的GFP的相应核苷酸序列完全一致,证明已构建含有GFP的重组麻疹病毒。
pT7MVS191、pT7MVS191-ATU-GFP的全长序列如SEQ ID NO.1和SEQ ID NO.2所示。
1.8构建pT7-MV-N、pT7-MV-P、pT7-MV-L
1.8.1pT7-MV-N的构建
pT7-MV-N的构建示意图如图5所示。
由于上游需要使用NcoI酶切位点，而N片段含有该位点，因此将N分为两段(1-67bp(BamHI)、68-1578bp)连入pT7-IRES His-C DNA载体。其中第一段为：合成1-67bp含有NcoI及BamHI粘性末端的两条单链(L1：5＇-cATGGCCACACTTTTAAGGAGCTTAGCATTGTTCAAAAGAA
ACAAGGACAAACCACCCATTACATCAG-3'；L2：5＇-cATGGCCACACTTTTAAGGAGCTTAG
CATTGTTCAAAAGAAACAAGGACAAACCACCCATTACATCAG-3')，经退火后互补，连入经NcoI+BamHI双酶切的pT7-IRES His-C DNA载体。第二段为：PCR扩增N片段68-1578(N-P1:5'–TCAGGATCCGGTGGAGCCATCA-3'；N-P2:5'-ggactagtCTAGTCTAGAAGATTTCTGTCAT
TG-3')片段，用BamHI、SpeI连入pT7-IRES His-C DNA载体。
pT7-MV-N的全长序列如SEQ ID NO.3所示。
1.8.2pT7-MV-P的构建
pT7-MV-P的构建示意图如图6所示。
设计引物(P-P1：5'-aaccatggCAGAAGAGCAGGCACGCC-3'；P-P2：5'-gctctagaCTACTTCATTATTATCTTCATCAGC-3')，扩增P基因，用NcoI、XbaI连入pT7-IRES His-CDNA载体。
pT7-MV-P的全长序列如SEQ ID NO.4所示。
1.8.3pT7-MV-L的构建
pT7-MV-L的构建示意图如图7所示。
L片段采用NcoI和SalI两个位点连入pT7-IRES His-C DNA载体。由于L片段含有NcoI切点，因此将L片段分两段(1-300bp(EcoRI)及剩下的部分)连入载体。扩增L片段第一段(L-P15’-cccatggATGGACTCGCTATCTGTCAACCAGA-3’；L-P2：5’-GTGAGTTAGTGTCCCTTAAG
CATTG-3’)，用NcoI、EcoRI连入pT7-IRES His-C DNA载体，L后部分是将PVAX-3’半分子下游酶切位点NotI改为SalI后，用EcoRI和SalI酶切新3’半分子，回收片段。最后将其连入构建好的pT7-L1载体。
pT7-MV-L的全长序列如SEQ ID NO.5所示。
综上，本发明构建得到了拯救麻疹病毒沪191毒株或表达GFP的重组麻疹病毒的系统所需的成分，本发明系统的示意图如图13所示。
1)含有麻疹病毒反基因组全长的质粒pT7MVS191(核苷酸序列如SEQ ID NO.1所示)，或者pT7MVS191-ATU-GFP(核苷酸序列如SEQ ID NO.2所示)；
2)辅助质粒1(pT7-MV-N，核苷酸序列如SEQ ID NO.3所示)，其含有麻疹病毒的核衣壳蛋白的编码基因；
3)辅助质粒2(pT7-MV-P，核苷酸序列如SEQ ID NO.4所示)，其含有麻疹病毒的磷蛋白的编码基因；
4)辅助质粒3(pT7-MV-L，核苷酸序列如SEQ ID NO.5所示)，其含有麻疹病毒的RNA聚合酶的编码基因。
实施例2采用本发明系统拯救麻疹病毒、表达GFP的重组麻疹病毒
采用本发明实施例1构建的载体拯救麻疹病毒、表达GFP的重组麻疹病毒：如图13所示，建立拯救系统。
1病毒的拯救
将BSR T7细胞于6孔板中37℃培养24h，待其长成单层，细胞汇合度为80％～90％时进行转染。12μg全长质粒pT7MVS191、12μgpT7MVS191-ATU-GFP分别和辅助质粒8μg pT7-MV-N，4μg pT7-MV-P，1μgpT7-MV-L混合，加入至100μL DMEM高糖培养液混匀后，再与已预混10μLLipofectamine 2000Reagent的100μL DMEM培养液混匀，室温放置30min后滴加至6孔板中，置37℃，5％CO2培养箱培养5-18h，更换新配制的含2％NBS的DMEM高糖培养液，24h培养。43.5℃热休克3h，继续培养3d。
吸取6孔板上清混合物感染Veroslam细胞，继续培养4d后，观察病变。收获冻融的病变细胞及上清混合物中的病毒颗粒，PCR鉴定拯救病毒。
2、检测
2.1细胞病变的检测结果
本实验共转染质粒接种至BSR T7细胞后，43.5℃热休克3h，以培养3d后的病毒上清感染Veroslam细胞，可观察到较为明显的CPE(细胞病变效应)(如图10，图11)，即有多核巨细胞，胞内和核内有嗜酸性包涵体，典型的合胞体样病变。用拯救的重组的pT7MVS191-ATU-GFP病毒感染Veroslam细胞，可在合胞体中观察到GFP的绿色荧光(如图11)。
2.2RT-PCR检测拯救病毒
P1代拯救病毒MV-S191在Veroslam细胞上连续传2代，待达到90％以上病变时后收获病毒液，提取细胞总RNA，以随机引物反转录合成cDNA，分别以3代病毒cDNA为扩增模板，以病毒基因MVF1、MVF4片段引物进行PCR扩增，MVF1片段扩增产物大小为：2044bp，MVF4片段扩增产物大小为：2330bp。
如图12所示，第1、2、3代毒种感染的Veroslam细胞可检测到病毒RNA存在。
3、本发明方法的拯救效率
重复步骤1的方法6次，检测本发明方法的拯救效率。
显微镜结果显示：pT7MVs191、pT7MVS191-ATU-GFP拯救上清与宿主细胞Veroslam共培养3d后，6组实验均可观察到细胞病变效应(可见合胞体)。荧光显微镜结果显示：pT7MVS191-ATU-GFP拯救上清与宿主细胞Veroslam共培养3d后，6组实验均可观察到细胞病变效应以及绿色荧光，这表明本发明成功拯救到麻疹病毒以及表达GFP的重组麻疹病毒。
共转染质粒量以及拯救结果如表2和表3所示：
表2：pT7MVs191拯救实验中转录质粒和3个辅助表达质粒的量及拯救结果
表3：pT7MVS191-ATU-GFP拯救实验中转录质粒和3个辅助表达质粒的量及拯救结果
由表2和表3可知，本发明的拯救系统以及方法共进行12次实验，显微镜观察及RT-PCR方法验证均成功收获具有感染性的麻疹病毒和重组麻疹病毒。说明该系统以及方法的拯救效率高，稳定性好。
综上，本发明构建得到了可以拯救麻疹病毒沪191毒株、表达GFP的重组麻疹病毒的系统，并且建立了拯救效率极高的拯救方法，可用来获得治疗性或预防性组合物中的麻疹病毒或重组麻疹病毒。
SEQUENCE LISTING
<110> 成都生物制品研究所有限责任公司
<120> 一种用于拯救麻疹病毒、重组麻疹病毒的系统以及方法
<130> GY014-17P1001
<160> 33
<170> PatentIn version 3.5
<210> 1
<211> 18695
<212> DNA
<213> pT7MVs191
<400> 1
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt gctagctagt tggcgcgcct aatacgactc actataacca 2280
aacaaagttg ggtaaggata gttcaatcaa tgatcatctt ctagtgcact taggattcaa 2340
gatcctatta tcagggacaa gagcaggatt agggatatcc gagatggcca cacttttaag 2400
gagcttagca ttgttcaaaa gaaacaagga caaaccaccc attacatcag gatccggtgg 2460
agccatcaga ggaatcaaac acattattat agtaccaatc cctggagatt cctcaattac 2520
cactcgatcc agacttctgg accggttggt caggttaatt ggaaacccgg atgtgagcgg 2580
gcccaaacta acaggggcac taataggtat attatcctta tttgtggagt ctccaggtca 2640
attgattcag aggatcaccg atgaccctga cgttagcata aggctgttag aggttgtcca 2700
gagtgaccag tcacaatctg gccttacctt cgcatcaaga ggtaccaaca tggaggatga 2760
ggcggaccaa tacttttcac atgatgatcc aattagtagt gatcaatcca ggttcggatg 2820
gtttgagaac aaggaaatct cagatattga agtgcaagac cctgagggat tcaacatgat 2880
tctgggtacc atcctagccc aaatttgggt cttgctcgca aaggcggtta cggccccaga 2940
cacggcagct gattcggagc taagaaggtg gataaagtac acccaacaaa gaagggtagt 3000
tggtgaattt agattggaga gaaaatggtt ggatgtggtg aggaacagga ttgccgagga 3060
cctctcctta cgccgattca tggtcgctct aatcctggat atcaagagaa cacccggaaa 3120
caaacccagg attgctgaaa tgatatgtga cattgataca tatatcgtag aggcaggatt 3180
agccagtttt atcctgacta ttaagtttgg gatagaaact atgtatcctg ctcttggact 3240
gcatgaattt gctggtgagt tatccacact tgagtccttg atgaaccttt accagcaaat 3300
gggggaaact gcaccctaca tggtaatcct ggagaactca attcagaaca agttcagtgc 3360
aggatcatac cctctgctct ggagctatgc catgggagta ggagtggaac ttgaaaactc 3420
catgggaggt ttgaactttg gccgatctta ctttgatcca gcatatttta gattagggca 3480
agagatggta aggaggtcag ctggaaaggt cagttccaca ttggcatctg aactcggtat 3540
cactgccgag gatgcaaggc ttgtttcaga gattgcaatg catactactg aggacaagat 3600
cagtagagcg gttggaccca gacaagccca agtatcattt ctacacggtg atcaaagtga 3660
gaatgagcta ccgagattgg ggggcaagga agacaggagg gtcaaacaga gtcgaggaga 3720
agccagggag agctacagag aaaccgggcc cagcagagca agtgatgcga gagccgccca 3780
tcttccaacc ggcacacccc cagacactga cactgcaccg gagtccagcc aagatccgca 3840
ggacagtcga aggtcagctg acgccctgct taggctgcaa gccatggcag gaatctcgga 3900
agaacaaggc tcagacacgg acacccccac agtgcacaat gacagaaatc ttctagacta 3960
ggtgcgagag gccgaggacc agaacaacat ccgcctaccc tccatcattg ttataaaaaa 4020
cttaggaacc aggtccacac agccgccagc ccatcaacca tccactccca cgattggagc 4080
cgatggcaga agagcaggca cgccatgtca aaaacggact ggaatgcatc cgggctctca 4140
aggccgagcc catcggctca ctggccatcg aggaagctat ggcagcatgg tcagaaatat 4200
cagacaaccc aggacaggag cgagccacct gcggggaaga gaaggcaggc agttcgggtc 4260
tcagcaaacc atgcctctca gcaattggat caactgaagg cggtgcacct cgcatccgcg 4320
gtcagggacc tggagagatc gatgacgacg ctgaaacttt gggaatcccc ccaagaaatc 4380
tccaggcatc aagcactggg ttacagtgtt attatgttta tgatcacagc ggtgaagcgg 4440
ttaagggaat ccaagatgct gactctatca tggttcaatc aggccttgat ggtgatagca 4500
ccctctcagg aggagacaat gaatctgaaa acagcgatgt ggatattggc gaacctgata 4560
ccgagggata tgctatcact gaccggggat ctgctcccat ctctatgggg ttcagggctt 4620
ctgatgttga aactgcagaa ggaggggaga tccacgagct cctgagactc caatccagag 4680
gcaacaactt tccgaagctt gggaaaactc tcaatgttcc tccgcccccg gaccccggta 4740
gggccagcac ttccgggaca cccattaaaa agggcacaga cgcgagatta gcctcatttg 4800
gaacggagat cgcgtcttta ttgacaggtg gtgcaaccca atgtgctcga aagtcaccct 4860
cggaaccatc agggccaggt gcacctgcgg ggaatgtccc cgagtgtgtg agcaatgccg 4920
cactgataca ggagtggaca cccgaatctg gtaccacaat ctccccgaga tcccagaata 4980
atgaagaagg gggagactat tatgatgatg agctgttctc tgatgtccaa gatattaaaa 5040
cagccttggc caaaatacac gaggataatc agaagataat ctccaagcta gaatcactgc 5100
tgttattgaa gggagaagtt gagtcaatta agaagcagat caacaggcaa aatatcagca 5160
tatccaccct ggaaggacac ctctcaagca tcatgatcgc cattcctgga cttgggaagg 5220
atcccaacga ccccactgca gatgtcgaaa tcaatcccga cttgaaaccc atcataggca 5280
gagattcagg ccgagcactg gccgaagttc tcaagaaacc cgttgccagc cgacaactcc 5340
aaggaatgac aaatggacgg accagttcca gaggacagct gctgaaggaa tttcagctaa 5400
agccgatcgg gaaaaagatg agctcagccg tcgggtttgt tcctgacacc ggccctgcat 5460
cacgcagtgt aatccgctcc attataaaat ccagccggct agaggaggat cggaagcgtt 5520
acctgatgac tctccttgat gatatcaaag gagccaatga tcttgccaag ttccaccaga 5580
tgctgatgaa gataataatg aagtagctac agctcaactt acctgccaac cccatgccag 5640
tcgacccaac tagtacaacc taaatccatt ataaaaaact taggagcaaa gtgattgcct 5700
cccaagttcc acaatgacag agatctacga cttcgacaag tcggcatggg acatcaaagg 5760
gtcgatcgct ccgatacaac ccaccaccta cagtgatggc aggctggtgc cccaggtcag 5820
agtcatagat cctggtctag gcgacaggaa ggatgaatgc tttatgtaca tgtttctgct 5880
gggggttgtt gaggacagcg atcccctagg gcctccaatc gggcgagcat ttgggtccct 5940
gcccttaggt gttggcagat ccacagcaaa gcccgaaaaa ctcctcaaag aggccactga 6000
gcttgacata gttgttagac gtacagcagg gctcaatgaa aaactggtgt tctacaacaa 6060
caccccacta actctcctca aaccttggag aaaggtccta acaacaggga gtgtcttcaa 6120
cgcaaaccaa gtgtgcaatg cggttaatct gataccgctc gataccccgc agaggttccg 6180
tgttgtttat atgagcatca cccgtctttc ggataacggg tattacaccg ttcctagaag 6240
aatgctggaa ttcagatcgg tcaatgcagt ggccttcaac ctgctggtga cccttaggat 6300
tgacaaggcg ataggccctg ggaagatcat cgacaataca gagcaacttc ctgaggcaac 6360
atttatggtc cacatcggga acttcaggag aaagaagagt gaagtctact ctgccgatta 6420
ttgcaaaatg aaaatcgaaa agatgggcct ggtttttgca cttggtggga tagggggcac 6480
cagtcttcac attagaagca caggcaaaat gagcaagact ctccatgcac aactcgggtt 6540
caagaagacc ttatgttacc cgctgatgga tatcaatgaa gaccttaatc gattactctg 6600
gaggagcaga tgcaagatag taagaatcca ggcagttttg cagccatcag ttcctcaaga 6660
attccgcatt tacgacgacg tgatcataaa tgatgaccaa ggactattca aagttctgta 6720
gaccgtagtg cccagcaatg cccgaaaacg acccccctca caatgacagc cagaaggccc 6780
ggacaaaaaa gccccctccg aaagactcca cggaccaagc gagaggccag ccagcagccg 6840
acggcaagcg cgaacaccag gcggccccag cacagaacag ccctgacaca aggccaccac 6900
cagccacccc aatctgcatc ctcctcgtgg gacccccgag gaccaacccc caaggctgcc 6960
cccgatccaa accaccaacc gcatccccac cacccccggg aaagaaaccc ccagcaactg 7020
gaaggcccct ccccctctcc ctcaacacaa gaactccaca accgaaccgc acaagcgacc 7080
gaggtgaccc aaccgcaggc atccgactcc ctagacagat cctctctccc cggcaaacta 7140
aacaaaactt agggccaagg aacatacaca cccaacagaa cccagacccc ggcccacggc 7200
gccgcgcccc caacccccga caaccagagg gagcccccaa ccaatcccgc cggctccccc 7260
ggtgcccaca ggcagtgaca ccaacccccg aacagaccca gcacccaacc atcgacaatc 7320
caagacgggg gggccccccc aaaaaaaggc ccccaggggc cgacagccag caccgcgagg 7380
aagcccaccc accccacaca cgaccacggc aaccaaacca gaacccagac caccctgggc 7440
caccagctcc cagactcggc catcaccccg cagaaaggaa aggccacaac ccgcgcaccc 7500
cagccccgat ccggcgggga gccacccaac ccgaaccagc acccaagagc gatccccgaa 7560
ggacccccga acagcaaagg acatcagtat cccacagcct ctccaagtcc cccggtctcc 7620
tcctcttctc gaagggacca aaagatcaat ccaccacacc cgacgacact caactcccca 7680
cccctaaagg agacaccggg aatcccagaa tcaagactca tccaatgtcc atcatgggtc 7740
tcaaggtgaa cgtctctgcc atattcatgg cagtactgtt aactctccaa acacccaccg 7800
gtcaaatcca ttggggcaat ctctctaaga taggggtggt aggaatagga agtgcaagct 7860
acaaagttat gactcgttcc agccatcaat cattagtcat aaaattaatg cccaatataa 7920
ctctcctcaa taactgcacg agggtagaga ttgcagaata caggagacta ctgagaacag 7980
ttttggaacc aattagagat gcacttaatg caatgaccca gaatataaga ccggttcaga 8040
gtgtagcttc aagtaggaga cacaagagat ttgcgggagt agtcctggca ggtgcggccc 8100
taggcgttgc cacagctgct cagataacag ccggcattgc acttcaccag tccatgctga 8160
actctcaagc catcgacaat ctgagagcga gcctggaaac tactaatcag gcaattgagg 8220
caatcagaca agcagggcag gagatgatat tggctgttca gggtgtccaa gactacatca 8280
ataatgagct gataccgtct atgaaccaac tatcttgtga tttaatcggc cagaagctcg 8340
ggctcaaatt gctcagatac tatacagaaa tcctgtcatt atttggcccc agcttacggg 8400
accccatatc tgcggagata tctatccagg ctttgagata tgcgcttgga ggagacatca 8460
ataaggtgtt agaaaagctc ggatacagtg gaggtgattt actgggcatc ttagagagca 8520
gaggaataaa ggcccggata actcacgtcg acacagagtc ctacttcatt gtcctcagta 8580
tagtctatcc gacgctgtcc gagattaagg gggtgattgt ccaccggcta gagggggtct 8640
cgtacaacat aggctctcaa gagtggtata ccactgtgcc caagtatgtt gcaacccaag 8700
ggtaccttat ctcgaatttt gatgagtcat cgtgtacttt catgccagag gggactgtgt 8760
gcagccaaaa tgccttgtac ccgatgagtc ctctgctcca agaatgcctc cgggggtcca 8820
ccaagtcctg tgctcgtaca ctcgtatccg ggtcttttgg gaaccggttc attttatcac 8880
aagggaacct aatagccaat tgtgcatcaa tcctttgcaa gtgttacaca acaggaacga 8940
tcattaatca agaccctgac aagatcctaa catacattgc tgccgatcac tgcccggtag 9000
tcgaggtgaa cggcgtgacc atccaagtcg ggagcaggag gtatccagac gctgtgtact 9060
tgcacagaat tgacctcggt cctcccatat cattggagag gttggacgta gggacaaatc 9120
tggggaatgc aattgctaag ttggaggatg ccaaggaact gttggagtca tcggaccaga 9180
tattgaggag tatgaaaggt ttatcgagca ctagcatagt ctacatcctg attgcagtgt 9240
gtcttggagg gttgataggg atccccgctt taatatgttg ctgcaggggg cgttgtaaca 9300
aaaagggaga acaagttggt atgtcaagac caggcctaaa gcctgatctt acgggaacat 9360
caaaatccta tgtaaggtcg ctctgatcct ctacaactct tgaaacacaa atgtcccaca 9420
agtctcctct tcgtcatcaa gcaaccaccg cacccagcat caagcccacc tgaaattatc 9480
tccggcttcc ctctggccga acaatatcgg tagttaatta aaacttaggg tgcaagatca 9540
tccacaatgt caccacaacg agaccggata aatgccttct acaaagataa cccccatccc 9600
aagggaagta ggatagtcat taacagagaa catcttatga ttgatagacc ttatgttttg 9660
ctggctgttc tgtttgtcat gtttctgagc ttgatcgggt tgctagccat tgcaggcatt 9720
agacttcatc gggcagccat ctacaccgca gagatccata aaagcctcag caccaatcta 9780
gatgtaacta actcaatcga gcatcaggtc aaggacgtgt tgacaccact cttcaaaatc 9840
atcggtgatg aagtgggcct gaggacacct cagagattca ctgacctagt gaaattcatc 9900
tctgacaaga ttaaattcct taatccggat agggagtacg acttaagaga tctcacttgg 9960
tgtatcaacc cgccagagag aatcaaattg gattatgatc aatactgtgc agatgtggct 10020
gctgaagagc tcatgaatgc attggtgaac tcaactctac tggagaccag aacaaccaat 10080
cagttcctag ctgtctcaaa gggaaactgc tcagggccca ctacaatcag aggtcaattc 10140
tcaaacatgt cgctgtccct gttagacttg tatttaggtc gaggttacaa tgtgtcatct 10200
atagtcacta tgacatccca gggaatgtat gggggaactt acctagtgga aaagcctaat 10260
ctgagcagca aaaggtcaga gttgtcacaa ctgagcatgt accgagtgtt tgaagtaggt 10320
gttatcagaa atccgggttt gggggctccg gtgttccata tgacaaacta tcttgagcaa 10380
ccagtcagta atgatctcag caactgtatg gtagctttgg gggagctcaa actcgcagcc 10440
ctttgtcacg gggaagattc tatcacaatt ccctatcagg gatcagggaa aggtgtcagc 10500
ttccagctcg tcaagctagg tgtctggaaa tccccaaccg acatgcaatc ctgggtcccc 10560
ttatcaacgg atgatccagt gatagacagg ctttacctct catctcacag aggtgttatc 10620
gctgacaatc aagcaaaatg ggctgtcccg acaacacgaa cagatgacaa gttgcgaatg 10680
gagacatgct tccaacaggc gtgtaagggt aaaatccaag cactctgcga gaatcccgag 10740
tgggcaccat tgaaggataa caggattcct tcatacgggg tcttgtctgt tgatctgagt 10800
ctgacagttg agcttaaaat caaaattgct tcgggattcg ggccattgat cacacacggt 10860
tcagggatgg acctatacaa atccaaccac aacaatgtgt attggctgac tatcccgcca 10920
atgaagaacc tagccttagg tgtaatcaac acattggagt ggataccgag attcaaggtt 10980
agtccctacc tcttcaatgt cccaattaag gaagcaggcg aagactgcca tgccccaaca 11040
tacctacctg cggaggtgga tgatgatgtc aaactcagtt ccaatctggt gattctacct 11100
ggtcaagatc tccaatatgt tttggcaacc tacgatactt ccagggttga acatgctgtg 11160
gtttattacg tttacagccc aagccgctca ttttcttact tttatccttt taggttgcct 11220
ataaaggggg tccccatcga attacaagtg gaatgcttca catgggacca aaaactctgg 11280
tgccgtcact tctgtgtgct tgcggactca gaatctggtg gacatatcac tcactctggg 11340
atggtgggca tgggagtcag ctgcacagtc acccgggaag atggaaccaa tcgcagatag 11400
ggctgctagt gaaccaatct catgatgtca cccatacatc aggcataccc actagtgtga 11460
aatagacatc agaattaaga aaaacgtagg gtccaagtgg ttccccgtta tggactcgct 11520
atctgtcaac cagatcttat accctgaagt tcacctagat agcccgatag ttaccaataa 11580
gatagtagcc atcctggagt atgctcgagt ccctcacgct tacagcctgg aggaccctac 11640
actgtgtcag aacatcaagc accgcctaaa aaacggattt tccaaccaaa tgattataaa 11700
caatgtggaa gttgggaatg tcatcaagtc caagcttagg agttatccgg cccactctca 11760
tattccatat ccaaattgta atcaggattt atttaacata gaagacaaag agtcaacgag 11820
gaagatccgt gaactcctca aaaaggggaa ttcgctgtac tccaaagtca gtgataaggt 11880
tttccaatgc ttaagggaca ctaactcacg gcttggccta ggctccgaat tgagggagga 11940
catcaaggag aaagttatta acttgggagt ttacatgcac agctcccagt ggtttgagcc 12000
ctttctgttt tggtttacag tcaagactga gatgaggtca gtgattaaat cacaaaccca 12060
tacttgccat aggaggagac acacacctgt attcttcact ggtagttcag ttgagttgct 12120
aatctctcgt gaccttgttg ctataatcag taaagagtct caacatgtat attacctgac 12180
atttgaactg gttttgatgt attgtgatgt catagagggg aggttaatga cagagaccgc 12240
tatgactatt gatgctaggt atacagagct tctaggaaga gtcagataca tgtggaaact 12300
gatagatggt ttcttccctg cactcgggaa tccaacttat caaattgtag ccatgctgga 12360
gcctctttca cttgcttacc tgcagctgag ggatataaca gtagaactca gaggtgcttt 12420
ccttaaccac tgctttactg aaatacatga tgttcttgac caaaacgggt tttctgatga 12480
aggtacttat catgagttaa ttgaagctct agattacatt ttcataactg atgacataca 12540
tctgacaggg gagattttct catttttcag aagtttcggc caccccagac ttgaagcagt 12600
aacggctgct gaaaatgtta ggaaatacat gaatcagcct aaagtcattg tgtatgagac 12660
tctgatgaaa ggtcatgcca tattttgtgg aatcataatc aacggctatc gtgacaggca 12720
cggaggcagt tggccaccgc tgaccctccc cctgcatgct gcagacacaa tccggaatgc 12780
tcaagcttca ggtgaagggt taacacatga gcagtgcgtt gataactgga aatcttttgc 12840
tggagtgaaa tttggctgct ttatgcctct tagcctggat agtgatctga caatgtacct 12900
aaaggacaag gcacttgctg ctctccaaag ggaatgggat tcagtttacc cgaaagagtt 12960
cctgcgttac gaccctccca agggaaccgg gtcacggagg cttgtagatg ttttccttaa 13020
tgattcgagc tttgacccat atgatgtgat aatgtatgtt gtaagtggag cttacctcca 13080
tgaccctgag ttcaacctgt cttacagcct gaaagaaaag gagatcaagg aaacaggtag 13140
actttttgct aaaatgactt acaaaatgag ggcatgccaa gtgattgctg aaaatctaat 13200
ctcaaacggg attggcaaat attttaagga caatgggatg gccaaggatg agcacgattt 13260
gactaaggca ctccacactc tagctgtctc aggagtcccc aaagatctca aagaaagtca 13320
cagggggggg ccagtcttaa aaacctactc ccgaagccca gtccacacaa gtaccaggaa 13380
cgtgagagca gcaaaagggt ttatagggtt ccctcaagta attcggcagg accaagacac 13440
tgatcatccg gagaatatgg aagcttacga gacagtcagt gcatttatca cgactgatct 13500
caagaagtac tgccttaatt ggagatatga gaccatcagc ttgtttgcac agaggctaaa 13560
tgagatttac ggattgccct cctttttcca gtggctgcat aagaggcttg agacctctgt 13620
cctgtatgta agtgaccctc attgcccccc cgaccttgac gcccatatcc cgttatataa 13680
agtccccaat gatcaaatct tcattaagta ccctatggga ggtatagaag ggtattgtca 13740
gaagctgtgg accatcagca ccattcccta tctatacctg gctgcttatg agagcggagt 13800
aaggattgct tcgttagtgc aaggggacaa tcagaccata gccgtaacaa aaagggtacc 13860
cagcacatgg ccctacaacc ttaagaaacg ggaagctgct agagtaacta gagattactt 13920
tgtaattctt aggcaaaggc tacatgatat tggccatcac ctcaaggcaa atgagacaat 13980
tgtttcatca catttttttg tctattcaaa aggaatatat tatgatgggc tacttgtgtc 14040
ccaatcactc aagagcatcg caagatgtgt attctggtca gagactatag ttgatgaaac 14100
aagggcagca tgcagtaata ttgctacaac aatggctaaa agcatcgaga gaggttatga 14160
ccgttacctt gcatattccc tgaacgtcct aaaagtgata cagcaaattc tgatctctct 14220
tggcttcaca atcaattcaa ccatgacccg ggatgtagtc atacccctcc tcacaaacaa 14280
cgacctctta ataaggatgg cactgttgcc cgctcctatt ggggggatga attatctgaa 14340
tatgagcagg ctgtttgtca gaaacatcgg tgatccagta acatcatcaa ttgctgatct 14400
caagagaatg attctcgcct cactaatgcc tgaagagacc ctccatcaag taatgacaca 14460
acaaccgggg gactcttcat tcctagactg ggctagcgac ccttactcag caaatcttgt 14520
atgtgtccag agcatcacta gactcctcaa gaacataact gcaaggtttg tcctgatcca 14580
tagcccaaac ccaatgttaa aaggattatt ccatgatgac agtaaagaag aggacgaggg 14640
actggcggca ttcctcatgg acaggcatat tatagtacct agggcagctc atgaaatcct 14700
ggatcatagt gtcacagggg caagagagtc tattgcaggc atgctggata ccacaaaagg 14760
cctgattcga gccagcatga ggaagggggg gttaacatct cgagtgataa ccagattgtc 14820
caattatgac tatgaacaat tcagagcagg gatggtgcta ttgacaggaa gaaagagaaa 14880
tgtcctcatt gacaaagagt catgttcagt gcagctggcg agagctctaa gaagccatat 14940
gtgggcgagg ctagctcgag gacggcctat ttacggcctt gaggtccctg atgtactaga 15000
atctatgcga ggccacctta ttcggcgtca tgagacatgt gtcatctgcg agtgtggatc 15060
agtcaactac ggatggtttt ttgtcccctc gggttgccaa ctggatgata ttgacaagga 15120
aacatcatcc ttgagagtcc catatattgg ttctaccact gatgagagaa cagacatgaa 15180
gcttgccttc gtaagagccc caagtcgatc cttgcgatct gctgttagaa tagcaacagt 15240
gtactcatgg gcttacggtg atgatgatag ctcttggaac gaagcctggt tgttggccag 15300
gcaaagggcc aatgtgagcc tggaggagct aagggtgatc actcccatct caacttcgac 15360
taatttagcg cataggttga gggatcgtag cactcaagtg aaatactcag gtacatccct 15420
tgtccgagtg gcgaggtata ccacaatctc caacgacaat ctctcatttg tcatatcaga 15480
taagaaggtt gatactaact ttatatacca acaaggaatg cttctagggt tgggtgtttt 15540
agaaacattg tttcgactcg agaaagatac cggatcatct aacacggtat tacatcttca 15600
cgtcgaaaca gattgttgcg tgatcccgat gatagatcat cccaggatac ccagctcccg 15660
caagctagag ctgagggcag agctatgtac caacccattg atatatgata atgcaccttt 15720
aattgacaga gatacaacaa ggctatacac ccagagccat aggaggcacc ttgtggaatt 15780
tgttacatgg tccacacccc aactatatca cattttagct aagtccacag cactatctat 15840
gattgacctg gtaacaaaat ttgagaagga ccatatgaat gaaatttcag ctctcatagg 15900
ggatgacgat atcaatagtt tcataactga gtttctgctc atagagccaa gattattcac 15960
tatctacttg ggccagtgtg cggccatcaa ttgggcattt gatgtacatt atcatagacc 16020
atcagggaaa tatcagatgg gtgagctgtt gtcatcgttc ctttctagaa tgagcaaagg 16080
agtgtttaag gtgcttgtca atgctctaag ccacccaaag atctacaaga aattctggca 16140
ttgtggtatt atagagccta tccatggtcc ttcacttgat gctcaaaact tgcacacaac 16200
tgtgtgcaac atggtttaca catgctatat gacctacctc gacctgttgt tgaatgaaga 16260
gttagaagag ttcacatttc tcttgtgtga aagcgacgag gatgtagtac cggacagatt 16320
cgacaacatc caggcaaaac acttatgtgt tctggcagat ttgtactgtc aaccagggac 16380
ctgcccacca attcgaggtc taagaccggt agagaaatgt gcagttctaa ccgaccatat 16440
caaggaagag gctaggttat ctccagcagg atcttcgtgg aacataaatc caattattgt 16500
agaccattac tcatgctctc tgacttatct ccggcgagga tcgatcaaac agataagatt 16560
gagagttgat ccaggattca ttttcgacgc cctcgctgag gtaaatgtca gtcagccaaa 16620
gatcggcagc aacaacatct caaatatgag catcaaggct ttcagacccc cacacgatga 16680
tgttgcaaaa ttgctcaaag atatcaacac aagcaagcac aatcttccca tttcaggggg 16740
caatctcgcc aattatgaaa tccatgcttt ccgcagaatc gggttgaact catctgcttg 16800
ctacaaagct gttgagatat caacattaat taggagatgc cttgagccag gggaggacgg 16860
cttgttcttg ggtgagggat cgggttctat gttgatcact tataaggaga tacttaaact 16920
aaacaagtgc ttctataata gtggggtttc cgccaattct agatctggtc aaagggaatt 16980
agcaccctat ccctccgaag ttggccttgt cgaacacaga atgggagtag gtaatattgt 17040
caaagtgctc tttaacggga ggcccgaagt cacgtgggta ggcagtgtag attgcttcaa 17100
tttcatagtt agtaatatcc ctacctctag tgtggggttt atccattcag atatagagac 17160
cttgcctaac aaagatacta tagagaagct agaggaattg gcagccatct tatcgatggc 17220
tctgctcctg ggcaaaatag gatcaatact ggtgattaag cttatgcctt tcagcgggga 17280
ttttgttcag ggatttataa gttatgtagg gtctcattat agagaagtga accttgtata 17340
ccctagatac agcaacttca tatctactga atcttatttg gttatgacag atctcaaggc 17400
taaccggcta atgaatcctg aaaagattaa gcagcagata attgaatcat ctgtgaggac 17460
ttcacctgga cttataggtc acatcctatc cattaagcaa ctaagctgca tacaagcaat 17520
tgtgggagac gcagttagta gaggtgatat caatcctact ctgaaaaaac ttacacctat 17580
agagcaggtg ctgatcaatt gcgggttggc aattaacgga cctaagctgt gcaaagaatt 17640
gatccaccat gatgttgcct cagggcaaga tggattgctt aattctatac tcatcctcta 17700
cagggagttg gcaagattca aagacaacca aagaagtcaa caagggatgt tccacgctta 17760
ccccgtattg gtaagtagca ggcaacgaga acttatatct aggatcaccc gcaaattttg 17820
ggggcacatt cttctttact ccgggaacag aaagttgata aataagttta tccagaatct 17880
caagtccggc tatctgatac tagacttaca ccagaatatc ttcgttaaga atctatccaa 17940
gtcagagaaa cagattatta tgacgggggg tttgaaacgc gagtgggttt ttaaggtaac 18000
agtcaaggag accaaagaat ggtataagtt agtcggatac agtgccctga ttaaggacta 18060
attggttgaa ctccggaacc ctaatcctgc cctaggtggt taggcattat ttgcaatata 18120
ttaaagaaaa ctttgaaaat acgaagtttc tattcccagc tttgtctggt ggccggcatg 18180
gtcatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 18240
aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgcggccgc 18300
taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 18360
taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 18420
cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt 18480
tctccttacg catctgtgcg gtatttcaca ccggtgcact ctcagtacaa tctgctctga 18540
tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 18600
ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 18660
tcagaggttt tcaccgtcat caccgaaacg cgcga 18695
<210> 2
<211> 19542
<212> DNA
<213> pT7MVS191-ATU-GFP
<400> 2
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt gctagctagt tggcgcgcct aatacgactc actataggaa 2280
agttgggtaa ggatagttca atcaatgatc atcttctagt gcacttagga ttcaagatcc 2340
tattatcagg gacaagagca ggattaggga tatccgagat ggccacactt ttaaggagct 2400
tagcattgtt caaaagaaac aaggacaaac cacccattac atcaggatcc ggtggagcca 2460
tcagaggaat caaacacatt attatagtac caatccctgg agattcctca attaccactc 2520
gatccagact tctggaccgg ttggtcaggt taattggaaa cccggatgtg agcgggccca 2580
aactaacagg ggcactaata ggtatattat ccttatttgt ggagtctcca ggtcaattga 2640
ttcagaggat caccgatgac cctgacgtta gcataaggct gttagaggtt gtccagagtg 2700
accagtcaca atctggcctt accttcgcat caagaggtac caacatggag gatgaggcgg 2760
accaatactt ttcacatgat gatccaatta gtagtgatca atccaggttc ggatggtttg 2820
agaacaagga aatctcagat attgaagtgc aagaccctga gggattcaac atgattctgg 2880
gtaccatcct agcccaaatt tgggtcttgc tcgcaaaggc ggttacggcc ccagacacgg 2940
cagctgattc ggagctaaga aggtggataa agtacaccca acaaagaagg gtagttggtg 3000
aatttagatt ggagagaaaa tggttggatg tggtgaggaa caggattgcc gaggacctct 3060
ccttacgccg attcatggtc gctctaatcc tggatatcaa gagaacaccc ggaaacaaac 3120
ccaggattgc tgaaatgata tgtgacattg atacatatat cgtagaggca ggattagcca 3180
gttttatcct gactattaag tttgggatag aaactatgta tcctgctctt ggactgcatg 3240
aatttgctgg tgagttatcc acacttgagt ccttgatgaa cctttaccag caaatggggg 3300
aaactgcacc ctacatggta atcctggaga actcaattca gaacaagttc agtgcaggat 3360
cataccctct gctctggagc tatgccatgg gagtaggagt ggaacttgaa aactccatgg 3420
gaggtttgaa ctttggccga tcttactttg atccagcata ttttagatta gggcaagaga 3480
tggtaaggag gtcagctgga aaggtcagtt ccacattggc atctgaactc ggtatcactg 3540
ccgaggatgc aaggcttgtt tcagagattg caatgcatac tactgaggac aagatcagta 3600
gagcggttgg acccagacaa gcccaagtat catttctaca cggtgatcaa agtgagaatg 3660
agctaccgag attggggggc aaggaagaca ggagggtcaa acagagtcga ggagaagcca 3720
gggagagcta cagagaaacc gggcccagca gagcaagtga tgcgagagcc gcccatcttc 3780
caaccggcac acccccagac actgacactg caccggagtc cagccaagat ccgcaggaca 3840
gtcgaaggtc agctgacgcc ctgcttaggc tgcaagccat ggcaggaatc tcggaagaac 3900
aaggctcaga cacggacacc cccacagtgc acaatgacag aaatcttcta gactaggtgc 3960
gagaggccga ggaccagaac aacatccgcc taccctccat cattgttata aaaaacttag 4020
gaaccaggtc cacacagccg ccagcccatc aaccatccac tcccacgatt ggagccgatg 4080
gcagaagagc aggcacgcca tgtcaaaaac ggactggaat gcatccgggc tctcaaggcc 4140
gagcccatcg gctcactggc catcgaggaa gctatggcag catggtcaga aatatcagac 4200
aacccaggac aggagcgagc cacctgcggg gaagagaagg caggcagttc gggtctcagc 4260
aaaccatgcc tctcagcaat tggatcaact gaaggcggtg cacctcgcat ccgcggtcag 4320
ggacctggag agatcgatga cgacgctgaa actttgggaa tccccccaag aaatctccag 4380
gcatcaagca ctgggttaca gtgttattat gtttatgatc acagcggtga agcggttaag 4440
ggaatccaag atgctgactc tatcatggtt caatcaggcc ttgatggtga tagcaccctc 4500
tcaggaggag acaatgaatc tgaaaacagc gatgtggata ttggcgaacc tgataccgag 4560
ggatatgcta tcactgaccg gggatctgct cccatctcta tggggttcag ggcttctgat 4620
gttgaaactg cagaaggagg ggagatccac gagctcctga gactccaatc cagaggcaac 4680
aactttccga agcttgggaa aactctcaat gttcctccgc ccccggaccc cggtagggcc 4740
agcacttccg ggacacccat taaaaagggc acagacgcga gattagcctc atttggaacg 4800
gagatcgcgt ctttattgac aggtggtgca acccaatgtg ctcgaaagtc accctcggaa 4860
ccatcagggc caggtgcacc tgcggggaat gtccccgagt gtgtgagcaa tgccgcactg 4920
atacaggagt ggacacccga atctggtacc acaatctccc cgagatccca gaataatgaa 4980
gaagggggag actattatga tgatgagctg ttctctgatg tccaagatat taaaacagcc 5040
ttggccaaaa tacacgagga taatcagaag ataatctcca agctagaatc actgctgtta 5100
ttgaagggag aagttgagtc aattaagaag cagatcaaca ggcaaaatat cagcatatcc 5160
accctggaag gacacctctc aagcatcatg atcgccattc ctggacttgg gaaggatccc 5220
aacgacccca ctgcagatgt cgaaatcaat cccgacttga aacccatcat aggcagagat 5280
tcaggccgag cactggccga agttctcaag aaacccgttg ccagccgaca actccaagga 5340
atgacaaatg gacggaccag ttccagagga cagctgctga aggaatttca gctaaagccg 5400
atcgggaaaa agatgagctc agccgtcggg tttgttcctg acaccggccc tgcatcacgc 5460
agtgtaatcc gctccattat aaaatccagc cggctagagg aggatcggaa gcgttacctg 5520
atgactctcc ttgatgatat caaaggagcc aatgatcttg ccaagttcca ccagatgctg 5580
atgaagataa taatgaagta gctacagctc aacttacctg ccaaccccat gccagtcgac 5640
ccaactagcc taccctccat cattgttata aaaaacttag gaaccaggtc cacacagccg 5700
ccagcccatc aacgcgtacg accatggtga gcaagcagat cctgaagaac accggcctgc 5760
aggagatcat gagcttcaag gtgaacctgg agggcgtggt gaacaaccac gtgttcacca 5820
tggagggctg cggcaagggc aacatcctgt tcggcaacca gctggtgcag atccgcgtga 5880
ccaagggcgc ccccctgccc ttcgccttcg acatcctgag ccccgccttc cagtacggca 5940
accgcacctt caccaagtac cccgaggaca tcagcgactt cttcatccag agcttccccg 6000
ccggcttcgt gtacgagcgc accctgcgct acgaggacgg cggcctggtg gagatccgca 6060
gcgacatcaa cctgatcgag gagatgttcg tgtaccgcgt ggagtacaag ggccgcaact 6120
tccccaacga cggccccgtg atgaagaaga ccatcaccgg cctgcagccc agcttcgagg 6180
tggtgtacat gaacgacggc gtgctggtgg gccaggtgat cctggtgtac cgcctgaaca 6240
gcggcaagtt ctacagctgc cacatgcgca ccctgatgaa gagcaagggc gtggtgaagg 6300
acttccccga gtaccacttc atccagcacc gcctggagaa gacctacgtg gaggacggcg 6360
gcttcgtgga gcagcacgag accgccatcg cccagctgac cagcctgggc aagcccctgg 6420
gcagcctgca cgagtgggtg taataggaat tcgatatcaa gtccaaaaaa acagctagct 6480
aagcgcgcag cgcttagacg tctcgcgact agtacaacct aaatccatta taaaaaactt 6540
aggagcaaag tgattgcctc ccaagttcca caatgacaga gatctacgac ttcgacaagt 6600
cggcatggga catcaaaggg tcgatcgctc cgatacaacc caccacctac agtgatggca 6660
ggctggtgcc ccaggtcaga gtcatagatc ctggtctagg cgacaggaag gatgaatgct 6720
ttatgtacat gtttctgctg ggggttgttg aggacagcga tcccctaggg cctccaatcg 6780
ggcgagcatt tgggtccctg cccttaggtg ttggcagatc cacagcaaag cccgaaaaac 6840
tcctcaaaga ggccactgag cttgacatag ttgttagacg tacagcaggg ctcaatgaaa 6900
aactggtgtt ctacaacaac accccactaa ctctcctcaa accttggaga aaggtcctaa 6960
caacagggag tgtcttcaac gcaaaccaag tgtgcaatgc ggttaatctg ataccgctcg 7020
ataccccgca gaggttccgt gttgtttata tgagcatcac ccgtctttcg gataacgggt 7080
attacaccgt tcctagaaga atgctggaat tcagatcggt caatgcagtg gccttcaacc 7140
tgctggtgac ccttaggatt gacaaggcga taggccctgg gaagatcatc gacaatacag 7200
agcaacttcc tgaggcaaca tttatggtcc acatcgggaa cttcaggaga aagaagagtg 7260
aagtctactc tgccgattat tgcaaaatga aaatcgaaaa gatgggcctg gtttttgcac 7320
ttggtgggat agggggcacc agtcttcaca ttagaagcac aggcaaaatg agcaagactc 7380
tccatgcaca actcgggttc aagaagacct tatgttaccc gctgatggat atcaatgaag 7440
accttaatcg attactctgg aggagcagat gcaagatagt aagaatccag gcagttttgc 7500
agccatcagt tcctcaagaa ttccgcattt acgacgacgt gatcataaat gatgaccaag 7560
gactattcaa agttctgtag accgtagtgc ccagcaatgc ccgaaaacga cccccctcac 7620
aatgacagcc agaaggcccg gacaaaaaag ccccctccga aagactccac ggaccaagcg 7680
agaggccagc cagcagccga cggcaagcgc gaacaccagg cggccccagc acagaacagc 7740
cctgacacaa ggccaccacc agccacccca atctgcatcc tcctcgtggg acccccgagg 7800
accaaccccc aaggctgccc ccgatccaaa ccaccaaccg catccccacc acccccggga 7860
aagaaacccc cagcaactgg aaggcccctc cccctctccc tcaacacaag aactccacaa 7920
ccgaaccgca caagcgaccg aggtgaccca accgcaggca tccgactccc tagacagatc 7980
ctctctcccc ggcaaactaa acaaaactta gggccaagga acatacacac ccaacagaac 8040
ccagaccccg gcccacggcg ccgcgccccc aacccccgac aaccagaggg agcccccaac 8100
caatcccgcc ggctcccccg gtgcccacag gcagtgacac caacccccga acagacccag 8160
cacccaacca tcgacaatcc aagacggggg ggccccccca aaaaaaggcc cccaggggcc 8220
gacagccagc accgcgagga agcccaccca ccccacacac gaccacggca accaaaccag 8280
aacccagacc accctgggcc accagctccc agactcggcc atcaccccgc agaaaggaaa 8340
ggccacaacc cgcgcacccc agccccgatc cggcggggag ccacccaacc cgaaccagca 8400
cccaagagcg atccccgaag gacccccgaa cagcaaagga catcagtatc ccacagcctc 8460
tccaagtccc ccggtctcct cctcttctcg aagggaccaa aagatcaatc caccacaccc 8520
gacgacactc aactccccac ccctaaagga gacaccggga atcccagaat caagactcat 8580
ccaatgtcca tcatgggtct caaggtgaac gtctctgcca tattcatggc agtactgtta 8640
actctccaaa cacccaccgg tcaaatccat tggggcaatc tctctaagat aggggtggta 8700
ggaataggaa gtgcaagcta caaagttatg actcgttcca gccatcaatc attagtcata 8760
aaattaatgc ccaatataac tctcctcaat aactgcacga gggtagagat tgcagaatac 8820
aggagactac tgagaacagt tttggaacca attagagatg cacttaatgc aatgacccag 8880
aatataagac cggttcagag tgtagcttca agtaggagac acaagagatt tgcgggagta 8940
gtcctggcag gtgcggccct aggcgttgcc acagctgctc agataacagc cggcattgca 9000
cttcaccagt ccatgctgaa ctctcaagcc atcgacaatc tgagagcgag cctggaaact 9060
actaatcagg caattgaggc aatcagacaa gcagggcagg agatgatatt ggctgttcag 9120
ggtgtccaag actacatcaa taatgagctg ataccgtcta tgaaccaact atcttgtgat 9180
ttaatcggcc agaagctcgg gctcaaattg ctcagatact atacagaaat cctgtcatta 9240
tttggcccca gcttacggga ccccatatct gcggagatat ctatccaggc tttgagatat 9300
gcgcttggag gagacatcaa taaggtgtta gaaaagctcg gatacagtgg aggtgattta 9360
ctgggcatct tagagagcag aggaataaag gcccggataa ctcacgtcga cacagagtcc 9420
tacttcattg tcctcagtat agtctatccg acgctgtccg agattaaggg ggtgattgtc 9480
caccggctag agggggtctc gtacaacata ggctctcaag agtggtatac cactgtgccc 9540
aagtatgttg caacccaagg gtaccttatc tcgaattttg atgagtcatc gtgtactttc 9600
atgccagagg ggactgtgtg cagccaaaat gccttgtacc cgatgagtcc tctgctccaa 9660
gaatgcctcc gggggtccac caagtcctgt gctcgtacac tcgtatccgg gtcttttggg 9720
aaccggttca ttttatcaca agggaaccta atagccaatt gtgcatcaat cctttgcaag 9780
tgttacacaa caggaacgat cattaatcaa gaccctgaca agatcctaac atacattgct 9840
gccgatcact gcccggtagt cgaggtgaac ggcgtgacca tccaagtcgg gagcaggagg 9900
tatccagacg ctgtgtactt gcacagaatt gacctcggtc ctcccatatc attggagagg 9960
ttggacgtag ggacaaatct ggggaatgca attgctaagt tggaggatgc caaggaactg 10020
ttggagtcat cggaccagat attgaggagt atgaaaggtt tatcgagcac tagcatagtc 10080
tacatcctga ttgcagtgtg tcttggaggg ttgataggga tccccgcttt aatatgttgc 10140
tgcagggggc gttgtaacaa aaagggagaa caagttggta tgtcaagacc aggcctaaag 10200
cctgatctta cgggaacatc aaaatcctat gtaaggtcgc tctgatcctc tacaactctt 10260
gaaacacaaa tgtcccacaa gtctcctctt cgtcatcaag caaccaccgc acccagcatc 10320
aagcccacct gaaattatct ccggcttccc tctggccgaa caatatcggt agttaattaa 10380
aacttagggt gcaagatcat ccacaatgtc accacaacga gaccggataa atgccttcta 10440
caaagataac ccccatccca agggaagtag gatagtcatt aacagagaac atcttatgat 10500
tgatagacct tatgttttgc tggctgttct gtttgtcatg tttctgagct tgatcgggtt 10560
gctagccatt gcaggcatta gacttcatcg ggcagccatc tacaccgcag agatccataa 10620
aagcctcagc accaatctag atgtaactaa ctcaatcgag catcaggtca aggacgtgtt 10680
gacaccactc ttcaaaatca tcggtgatga agtgggcctg aggacacctc agagattcac 10740
tgacctagtg aaattcatct ctgacaagat taaattcctt aatccggata gggagtacga 10800
cttaagagat ctcacttggt gtatcaaccc gccagagaga atcaaattgg attatgatca 10860
atactgtgca gatgtggctg ctgaagagct catgaatgca ttggtgaact caactctact 10920
ggagaccaga acaaccaatc agttcctagc tgtctcaaag ggaaactgct cagggcccac 10980
tacaatcaga ggtcaattct caaacatgtc gctgtccctg ttagacttgt atttaggtcg 11040
aggttacaat gtgtcatcta tagtcactat gacatcccag ggaatgtatg ggggaactta 11100
cctagtggaa aagcctaatc tgagcagcaa aaggtcagag ttgtcacaac tgagcatgta 11160
ccgagtgttt gaagtaggtg ttatcagaaa tccgggtttg ggggctccgg tgttccatat 11220
gacaaactat cttgagcaac cagtcagtaa tgatctcagc aactgtatgg tagctttggg 11280
ggagctcaaa ctcgcagccc tttgtcacgg ggaagattct atcacaattc cctatcaggg 11340
atcagggaaa ggtgtcagct tccagctcgt caagctaggt gtctggaaat ccccaaccga 11400
catgcaatcc tgggtcccct tatcaacgga tgatccagtg atagacaggc tttacctctc 11460
atctcacaga ggtgttatcg ctgacaatca agcaaaatgg gctgtcccga caacacgaac 11520
agatgacaag ttgcgaatgg agacatgctt ccaacaggcg tgtaagggta aaatccaagc 11580
actctgcgag aatcccgagt gggcaccatt gaaggataac aggattcctt catacggggt 11640
cttgtctgtt gatctgagtc tgacagttga gcttaaaatc aaaattgctt cgggattcgg 11700
gccattgatc acacacggtt cagggatgga cctatacaaa tccaaccaca acaatgtgta 11760
ttggctgact atcccgccaa tgaagaacct agccttaggt gtaatcaaca cattggagtg 11820
gataccgaga ttcaaggtta gtccctacct cttcaatgtc ccaattaagg aagcaggcga 11880
agactgccat gccccaacat acctacctgc ggaggtggat gatgatgtca aactcagttc 11940
caatctggtg attctacctg gtcaagatct ccaatatgtt ttggcaacct acgatacttc 12000
cagggttgaa catgctgtgg tttattacgt ttacagccca agccgctcat tttcttactt 12060
ttatcctttt aggttgccta taaagggggt ccccatcgaa ttacaagtgg aatgcttcac 12120
atgggaccaa aaactctggt gccgtcactt ctgtgtgctt gcggactcag aatctggtgg 12180
acatatcact cactctggga tggtgggcat gggagtcagc tgcacagtca cccgggaaga 12240
tggaaccaat cgcagatagg gctgctagtg aaccaatctc atgatgtcac ccatacatca 12300
ggcataccca ctagtgtgaa atagacatca gaattaagaa aaacgtaggg tccaagtggt 12360
tccccgttat ggactcgcta tctgtcaacc agatcttata ccctgaagtt cacctagata 12420
gcccgatagt taccaataag atagtagcca tcctggagta tgctcgagtc cctcacgctt 12480
acagcctgga ggaccctaca ctgtgtcaga acatcaagca ccgcctaaaa aacggatttt 12540
ccaaccaaat gattataaac aatgtggaag ttgggaatgt catcaagtcc aagcttagga 12600
gttatccggc ccactctcat attccatatc caaattgtaa tcaggattta tttaacatag 12660
aagacaaaga gtcaacgagg aagatccgtg aactcctcaa aaaggggaat tcgctgtact 12720
ccaaagtcag tgataaggtt ttccaatgct taagggacac taactcacgg cttggcctag 12780
gctccgaatt gagggaggac atcaaggaga aagttattaa cttgggagtt tacatgcaca 12840
gctcccagtg gtttgagccc tttctgtttt ggtttacagt caagactgag atgaggtcag 12900
tgattaaatc acaaacccat acttgccata ggaggagaca cacacctgta ttcttcactg 12960
gtagttcagt tgagttgcta atctctcgtg accttgttgc tataatcagt aaagagtctc 13020
aacatgtata ttacctgaca tttgaactgg ttttgatgta ttgtgatgtc atagagggga 13080
ggttaatgac agagaccgct atgactattg atgctaggta tacagagctt ctaggaagag 13140
tcagatacat gtggaaactg atagatggtt tcttccctgc actcgggaat ccaacttatc 13200
aaattgtagc catgctggag cctctttcac ttgcttacct gcagctgagg gatataacag 13260
tagaactcag aggtgctttc cttaaccact gctttactga aatacatgat gttcttgacc 13320
aaaacgggtt ttctgatgaa ggtacttatc atgagttaat tgaagctcta gattacattt 13380
tcataactga tgacatacat ctgacagggg agattttctc atttttcaga agtttcggcc 13440
accccagact tgaagcagta acggctgctg aaaatgttag gaaatacatg aatcagccta 13500
aagtcattgt gtatgagact ctgatgaaag gtcatgccat attttgtgga atcataatca 13560
acggctatcg tgacaggcac ggaggcagtt ggccaccgct gaccctcccc ctgcatgctg 13620
cagacacaat ccggaatgct caagcttcag gtgaagggtt aacacatgag cagtgcgttg 13680
ataactggaa atcttttgct ggagtgaaat ttggctgctt tatgcctctt agcctggata 13740
gtgatctgac aatgtaccta aaggacaagg cacttgctgc tctccaaagg gaatgggatt 13800
cagtttaccc gaaagagttc ctgcgttacg accctcccaa gggaaccggg tcacggaggc 13860
ttgtagatgt tttccttaat gattcgagct ttgacccata tgatgtgata atgtatgttg 13920
taagtggagc ttacctccat gaccctgagt tcaacctgtc ttacagcctg aaagaaaagg 13980
agatcaagga aacaggtaga ctttttgcta aaatgactta caaaatgagg gcatgccaag 14040
tgattgctga aaatctaatc tcaaacggga ttggcaaata ttttaaggac aatgggatgg 14100
ccaaggatga gcacgatttg actaaggcac tccacactct agctgtctca ggagtcccca 14160
aagatctcaa agaaagtcac aggggggggc cagtcttaaa aacctactcc cgaagcccag 14220
tccacacaag taccaggaac gtgagagcag caaaagggtt tatagggttc cctcaagtaa 14280
ttcggcagga ccaagacact gatcatccgg agaatatgga agcttacgag acagtcagtg 14340
catttatcac gactgatctc aagaagtact gccttaattg gagatatgag accatcagct 14400
tgtttgcaca gaggctaaat gagatttacg gattgccctc ctttttccag tggctgcata 14460
agaggcttga gacctctgtc ctgtatgtaa gtgaccctca ttgccccccc gaccttgacg 14520
cccatatccc gttatataaa gtccccaatg atcaaatctt cattaagtac cctatgggag 14580
gtatagaagg gtattgtcag aagctgtgga ccatcagcac cattccctat ctatacctgg 14640
ctgcttatga gagcggagta aggattgctt cgttagtgca aggggacaat cagaccatag 14700
ccgtaacaaa aagggtaccc agcacatggc cctacaacct taagaaacgg gaagctgcta 14760
gagtaactag agattacttt gtaattctta ggcaaaggct acatgatatt ggccatcacc 14820
tcaaggcaaa tgagacaatt gtttcatcac atttttttgt ctattcaaaa ggaatatatt 14880
atgatgggct acttgtgtcc caatcactca agagcatcgc aagatgtgta ttctggtcag 14940
agactatagt tgatgaaaca agggcagcat gcagtaatat tgctacaaca atggctaaaa 15000
gcatcgagag aggttatgac cgttaccttg catattccct gaacgtccta aaagtgatac 15060
agcaaattct gatctctctt ggcttcacaa tcaattcaac catgacccgg gatgtagtca 15120
tacccctcct cacaaacaac gacctcttaa taaggatggc actgttgccc gctcctattg 15180
gggggatgaa ttatctgaat atgagcaggc tgtttgtcag aaacatcggt gatccagtaa 15240
catcatcaat tgctgatctc aagagaatga ttctcgcctc actaatgcct gaagagaccc 15300
tccatcaagt aatgacacaa caaccggggg actcttcatt cctagactgg gctagcgacc 15360
cttactcagc aaatcttgta tgtgtccaga gcatcactag actcctcaag aacataactg 15420
caaggtttgt cctgatccat agcccaaacc caatgttaaa aggattattc catgatgaca 15480
gtaaagaaga ggacgaggga ctggcggcat tcctcatgga caggcatatt atagtaccta 15540
gggcagctca tgaaatcctg gatcatagtg tcacaggggc aagagagtct attgcaggca 15600
tgctggatac cacaaaaggc ctgattcgag ccagcatgag gaaggggggg ttaacatctc 15660
gagtgataac cagattgtcc aattatgact atgaacaatt cagagcaggg atggtgctat 15720
tgacaggaag aaagagaaat gtcctcattg acaaagagtc atgttcagtg cagctggcga 15780
gagctctaag aagccatatg tgggcgaggc tagctcgagg acggcctatt tacggccttg 15840
aggtccctga tgtactagaa tctatgcgag gccaccttat tcggcgtcat gagacatgtg 15900
tcatctgcga gtgtggatca gtcaactacg gatggttttt tgtcccctcg ggttgccaac 15960
tggatgatat tgacaaggaa acatcatcct tgagagtccc atatattggt tctaccactg 16020
atgagagaac agacatgaag cttgccttcg taagagcccc aagtcgatcc ttgcgatctg 16080
ctgttagaat agcaacagtg tactcatggg cttacggtga tgatgatagc tcttggaacg 16140
aagcctggtt gttggccagg caaagggcca atgtgagcct ggaggagcta agggtgatca 16200
ctcccatctc aacttcgact aatttagcgc ataggttgag ggatcgtagc actcaagtga 16260
aatactcagg tacatccctt gtccgagtgg cgaggtatac cacaatctcc aacgacaatc 16320
tctcatttgt catatcagat aagaaggttg atactaactt tatataccaa caaggaatgc 16380
ttctagggtt gggtgtttta gaaacattgt ttcgactcga gaaagatacc ggatcatcta 16440
acacggtatt acatcttcac gtcgaaacag attgttgcgt gatcccgatg atagatcatc 16500
ccaggatacc cagctcccgc aagctagagc tgagggcaga gctatgtacc aacccattga 16560
tatatgataa tgcaccttta attgacagag atacaacaag gctatacacc cagagccata 16620
ggaggcacct tgtggaattt gttacatggt ccacacccca actatatcac attttagcta 16680
agtccacagc actatctatg attgacctgg taacaaaatt tgagaaggac catatgaatg 16740
aaatttcagc tctcataggg gatgacgata tcaatagttt cataactgag tttctgctca 16800
tagagccaag attattcact atctacttgg gccagtgtgc ggccatcaat tgggcatttg 16860
atgtacatta tcatagacca tcagggaaat atcagatggg tgagctgttg tcatcgttcc 16920
tttctagaat gagcaaagga gtgtttaagg tgcttgtcaa tgctctaagc cacccaaaga 16980
tctacaagaa attctggcat tgtggtatta tagagcctat ccatggtcct tcacttgatg 17040
ctcaaaactt gcacacaact gtgtgcaaca tggtttacac atgctatatg acctacctcg 17100
acctgttgtt gaatgaagag ttagaagagt tcacatttct cttgtgtgaa agcgacgagg 17160
atgtagtacc ggacagattc gacaacatcc aggcaaaaca cttatgtgtt ctggcagatt 17220
tgtactgtca accagggacc tgcccaccaa ttcgaggtct aagaccggta gagaaatgtg 17280
cagttctaac cgaccatatc aaggaagagg ctaggttatc tccagcagga tcttcgtgga 17340
acataaatcc aattattgta gaccattact catgctctct gacttatctc cggcgaggat 17400
cgatcaaaca gataagattg agagttgatc caggattcat tttcgacgcc ctcgctgagg 17460
taaatgtcag tcagccaaag atcggcagca acaacatctc aaatatgagc atcaaggctt 17520
tcagaccccc acacgatgat gttgcaaaat tgctcaaaga tatcaacaca agcaagcaca 17580
atcttcccat ttcagggggc aatctcgcca attatgaaat ccatgctttc cgcagaatcg 17640
ggttgaactc atctgcttgc tacaaagctg ttgagatatc aacattaatt aggagatgcc 17700
ttgagccagg ggaggacggc ttgttcttgg gtgagggatc gggttctatg ttgatcactt 17760
ataaggagat acttaaacta aacaagtgct tctataatag tggggtttcc gccaattcta 17820
gatctggtca aagggaatta gcaccctatc cctccgaagt tggccttgtc gaacacagaa 17880
tgggagtagg taatattgtc aaagtgctct ttaacgggag gcccgaagtc acgtgggtag 17940
gcagtgtaga ttgcttcaat ttcatagtta gtaatatccc tacctctagt gtggggttta 18000
tccattcaga tatagagacc ttgcctaaca aagatactat agagaagcta gaggaattgg 18060
cagccatctt atcgatggct ctgctcctgg gcaaaatagg atcaatactg gtgattaagc 18120
ttatgccttt cagcggggat tttgttcagg gatttataag ttatgtaggg tctcattata 18180
gagaagtgaa ccttgtatac cctagataca gcaacttcat atctactgaa tcttatttgg 18240
ttatgacaga tctcaaggct aaccggctaa tgaatcctga aaagattaag cagcagataa 18300
ttgaatcatc tgtgaggact tcacctggac ttataggtca catcctatcc attaagcaac 18360
taagctgcat acaagcaatt gtgggagacg cagttagtag aggtgatatc aatcctactc 18420
tgaaaaaact tacacctata gagcaggtgc tgatcaattg cgggttggca attaacggac 18480
ctaagctgtg caaagaattg atccaccatg atgttgcctc agggcaagat ggattgctta 18540
attctatact catcctctac agggagttgg caagattcaa agacaaccaa agaagtcaac 18600
aagggatgtt ccacgcttac cccgtattgg taagtagcag gcaacgagaa cttatatcta 18660
ggatcacccg caaattttgg gggcacattc ttctttactc cgggaacaga aagttgataa 18720
ataagtttat ccagaatctc aagtccggct atctgatact agacttacac cagaatatct 18780
tcgttaagaa tctatccaag tcagagaaac agattattat gacggggggt ttgaaacgcg 18840
agtgggtttt taaggtaaca gtcaaggaga ccaaagaatg gtataagtta gtcggataca 18900
gtgccctgat taaggactaa ttggttgaac tccggaaccc taatcctgcc ctaggtggtt 18960
aggcattatt tgcaatatat taaagaaaac tttgaaaata cgaagtttct attcccagct 19020
ttgtctggtg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 19080
gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 19140
cggccgctaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 19200
cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 19260
cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc 19320
ggtattttct ccttacgcat ctgtgcggta tttcacaccg gtgcactctc agtacaatct 19380
gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 19440
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 19500
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc ga 19542
<210> 3
<211> 4968
<212> DNA
<213> pT7-MV-N
<400> 3
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt taatacgact cactataggc aaccatgata acgttactgg 2280
ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 2340
gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 2400
taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 2460
agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 2520
gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 2580
tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 2640
atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 2700
tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 2760
aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2820
tatggccaca accatggcca cacttttaag gagcttagca ttgttcaaaa gaaacaagga 2880
caaaccaccc attacatcag gatccggtgg agccatcaga ggaatcaaac acattattat 2940
agtaccaatc cctggagatt cctcaattac cactcgatcc agacttctgg accggttggt 3000
caggttaatt ggaaacccgg atgtgagcgg gcccaaacta acaggggcac taataggtat 3060
attatcctta tttgtggagt ctccaggtca attgattcag aggatcaccg atgaccctga 3120
cgttagcata aggctgttag aggttgtcca gagtgaccag tcacaatctg gccttacctt 3180
cgcatcaaga ggtaccaaca tggaggatga ggcggaccaa tacttttcac atgatgatcc 3240
aattagtagt gatcaatcca ggttcggatg gtttgagaac aaggaaatct cagatattga 3300
agtgcaagac cctgagggat tcaacatgat tctgggtacc atcctagccc aaatttgggt 3360
cttgctcgca aaggcggtta cggccccaga cacggcagct gattcggagc taagaaggtg 3420
gataaagtac acccaacaaa gaagggtagt tggtgaattt agattggaga gaaaatggtt 3480
ggatgtggtg aggaacagga ttgccgagga cctctcctta cgccgattca tggtcgctct 3540
aatcctggat atcaagagaa cacccggaaa caaacccagg attgctgaaa tgatatgtga 3600
cattgataca tatatcgtag aggcaggatt agccagtttt atcctgacta ttaagtttgg 3660
gatagaaact atgtatcctg ctcttggact gcatgaattt gctggtgagt tatccacact 3720
tgagtccttg atgaaccttt accagcaaat gggggaaact gcaccctaca tggtaatcct 3780
ggagaactca attcagaaca agttcagtgc aggatcatac cctctgctct ggagctatgc 3840
catgggagta ggagtggaac ttgaaaactc catgggaggt ttgaactttg gccgatctta 3900
ctttgatcca gcatatttta gattagggca agagatggta aggaggtcag ctggaaaggt 3960
cagttccaca ttggcatctg aactcggtat cactgccgag gatgcaaggc ttgtttcaga 4020
gattgcaatg catactactg aggacaagat cagtagagcg gttggaccca gacaagccca 4080
agtatcattt ctacacggtg atcaaagtga gaatgagcta ccgagattgg ggggcaagga 4140
agacaggagg gtcaaacaga gtcgaggaga agccagggag agctacagag aaaccgggcc 4200
cagcagagca agtgatgcga gagccgccca tcttccaacc ggcacacccc cagacactga 4260
cactgcaccg gagtccagcc aagatccgca ggacagtcga aggtcagctg acgccctgct 4320
taggctgcaa gccatggcag gaatctcgga agaacaaggc tcagacacgg acacccccac 4380
agtgcacaat gacagaaatc ttctagacta gactagtgtc gacctgcagt ctagaatcga 4440
agggcgccac catcaccatc accattaggt aatctctgaa aaaaaaaaaa aaaaaaaaaa 4500
aaaaaaaaac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttggat 4560
catccgcggc cgctaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 4620
gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 4680
aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 4740
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccggtgc actctcagta 4800
caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 4860
cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 4920
ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga 4968
<210> 4
<211> 4896
<212> DNA
<213> pT7-MV-P
<400> 4
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt taatacgact cactataggc aaccatgata acgttactgg 2280
ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 2340
gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 2400
taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 2460
agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 2520
gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 2580
tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 2640
atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 2700
tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 2760
aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2820
tatggccaca accatggcag aagagcaggc acgccatgtc aaaaacggac tggaatgcat 2880
ccgggctctc aaggccgagc ccatcggctc actggccatc gaggaagcta tggcagcatg 2940
gtcagaaata tcagacaacc caggacagga gcgagccacc tgcggggaag agaaggcagg 3000
cagttcgggt ctcagcaaac catgcctctc agcaattgga tcaactgaag gcggtgcacc 3060
tcgcatccgc ggtcagggac ctggagagat cgatgacgac gctgaaactt tgggaatccc 3120
cccaagaaat ctccaggcat caagcactgg gttacagtgt tattatgttt atgatcacag 3180
cggtgaagcg gttaagggaa tccaagatgc tgactctatc atggttcaat caggccttga 3240
tggtgatagc accctctcag gaggagacaa tgaatctgaa aacagcgatg tggatattgg 3300
cgaacctgat accgagggat atgctatcac tgaccgggga tctgctccca tctctatggg 3360
gttcagggct tctgatgttg aaactgcaga aggaggggag atccacgagc tcctgagact 3420
ccaatccaga ggcaacaact ttccgaagct tgggaaaact ctcaatgttc ctccgccccc 3480
ggaccccggt agggccagca cttccgggac acccattaaa aagggcacag acgcgagatt 3540
agcctcattt ggaacggaga tcgcgtcttt attgacaggt ggtgcaaccc aatgtgctcg 3600
aaagtcaccc tcggaaccat cagggccagg tgcacctgcg gggaatgtcc ccgagtgtgt 3660
gagcaatgcc gcactgatac aggagtggac acccgaatct ggtaccacaa tctccccgag 3720
atcccagaat aatgaagaag ggggagacta ttatgatgat gagctgttct ctgatgtcca 3780
agatattaaa acagccttgg ccaaaataca cgaggataat cagaagataa tctccaagct 3840
agaatcactg ctgttattga agggagaagt tgagtcaatt aagaagcaga tcaacaggca 3900
aaatatcagc atatccaccc tggaaggaca cctctcaagc atcatgatcg ccattcctgg 3960
acttgggaag gatcccaacg accccactgc agatgtcgaa atcaatcccg acttgaaacc 4020
catcataggc agagattcag gccgagcact ggccgaagtt ctcaagaaac ccgttgccag 4080
ccgacaactc caaggaatga caaatggacg gaccagttcc agaggacagc tgctgaagga 4140
atttcagcta aagccgatcg ggaaaaagat gagctcagcc gtcgggtttg ttcctgacac 4200
cggccctgca tcacgcagtg taatccgctc cattataaaa tccagccggc tagaggagga 4260
tcggaagcgt tacctgatga ctctccttga tgatatcaaa ggagccaatg atcttgccaa 4320
gttccaccag atgctgatga agataataat gaagtagtct agaatcgaag ggcgccacca 4380
tcaccatcac cattaggtaa tctctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaacta 4440
gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttggatca tccgcggccg 4500
ctaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 4560
ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 4620
ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt 4680
ttctccttac gcatctgtgc ggtatttcac accggtgcac tctcagtaca atctgctctg 4740
atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4800
cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4860
gtcagaggtt ttcaccgtca tcaccgaaac gcgcga 4896
<210> 5
<211> 9945
<212> DNA
<213> pT7-MV-L
<400> 5
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt taatacgact cactataggc aaccatgata acgttactgg 2280
ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 2340
gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 2400
taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 2460
agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 2520
gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 2580
tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 2640
atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg 2700
tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa 2760
aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa 2820
tatggccaca accatggatg gactcgctat ctgtcaacca gatcttatac cctgaagttc 2880
acctagatag cccgatagtt accaataaga tagtagccat cctggagtat gctcgagtcc 2940
ctcacgctta cagcctggag gaccctacac tgtgtcagaa catcaagcac cgcctaaaaa 3000
acggattttc caaccaaatg attataaaca atgtggaagt tgggaatgtc atcaagtcca 3060
agcttaggag ttatccggcc cactctcata ttccatatcc aaattgtaat caggatttat 3120
ttaacataga agacaaagag tcaacgagga agatccgtga actcctcaaa aaggggaatt 3180
cgctgtactc caaagtcagt gataaggttt tccaatgctt aagggacact aactcacggc 3240
ttggcctagg ctccgaattg agggaggaca tcaaggagaa agttattaac ttgggagttt 3300
acatgcacag ctcccagtgg tttgagccct ttctgttttg gtttacagtc aagactgaga 3360
tgaggtcagt gattaaatca caaacccata cttgccatag gaggagacac acacctgtat 3420
tcttcactgg tagttcagtt gagttgctaa tctctcgtga ccttgttgct ataatcagta 3480
aagagtctca acatgtatat tacctgacat ttgaactggt tttgatgtat tgtgatgtca 3540
tagaggggag gttaatgaca gagaccgcta tgactattga tgctaggtat acagagcttc 3600
taggaagagt cagatacatg tggaaactga tagatggttt cttccctgca ctcgggaatc 3660
caacttatca aattgtagcc atgctggagc ctctttcact tgcttacctg cagctgaggg 3720
atataacagt agaactcaga ggtgctttcc ttaaccactg ctttactgaa atacatgatg 3780
ttcttgacca aaacgggttt tctgatgaag gtacttatca tgagttaatt gaagctctag 3840
attacatttt cataactgat gacatacatc tgacagggga gattttctca tttttcagaa 3900
gtttcggcca ccccagactt gaagcagtaa cggctgctga aaatgttagg aaatacatga 3960
atcagcctaa agtcattgtg tatgagactc tgatgaaagg tcatgccata ttttgtggaa 4020
tcataatcaa cggctatcgt gacaggcacg gaggcagttg gccaccgctg accctccccc 4080
tgcatgctgc agacacaatc cggaatgctc aagcttcagg tgaagggtta acacatgagc 4140
agtgcgttga taactggaaa tcttttgctg gagtgaaatt tggctgcttt atgcctctta 4200
gcctggatag tgatctgaca atgtacctaa aggacaaggc acttgctgct ctccaaaggg 4260
aatgggattc agtttacccg aaagagttcc tgcgttacga ccctcccaag ggaaccgggt 4320
cacggaggct tgtagatgtt ttccttaatg attcgagctt tgacccatat gatgtgataa 4380
tgtatgttgt aagtggagct tacctccatg accctgagtt caacctgtct tacagcctga 4440
aagaaaagga gatcaaggaa acaggtagac tttttgctaa aatgacttac aaaatgaggg 4500
catgccaagt gattgctgaa aatctaatct caaacgggat tggcaaatat tttaaggaca 4560
atgggatggc caaggatgag cacgatttga ctaaggcact ccacactcta gctgtctcag 4620
gagtccccaa agatctcaaa gaaagtcaca ggggggggcc agtcttaaaa acctactccc 4680
gaagcccagt ccacacaagt accaggaacg tgagagcagc aaaagggttt atagggttcc 4740
ctcaagtaat tcggcaggac caagacactg atcatccgga gaatatggaa gcttacgaga 4800
cagtcagtgc atttatcacg actgatctca agaagtactg ccttaattgg agatatgaga 4860
ccatcagctt gtttgcacag aggctaaatg agatttacgg attgccctcc tttttccagt 4920
ggctgcataa gaggcttgag acctctgtcc tgtatgtaag tgaccctcat tgcccccccg 4980
accttgacgc ccatatcccg ttatataaag tccccaatga tcaaatcttc attaagtacc 5040
ctatgggagg tatagaaggg tattgtcaga agctgtggac catcagcacc attccctatc 5100
tatacctggc tgcttatgag agcggagtaa ggattgcttc gttagtgcaa ggggacaatc 5160
agaccatagc cgtaacaaaa agggtaccca gcacatggcc ctacaacctt aagaaacggg 5220
aagctgctag agtaactaga gattactttg taattcttag gcaaaggcta catgatattg 5280
gccatcacct caaggcaaat gagacaattg tttcatcaca tttttttgtc tattcaaaag 5340
gaatatatta tgatgggcta cttgtgtccc aatcactcaa gagcatcgca agatgtgtat 5400
tctggtcaga gactatagtt gatgaaacaa gggcagcatg cagtaatatt gctacaacaa 5460
tggctaaaag catcgagaga ggttatgacc gttaccttgc atattccctg aacgtcctaa 5520
aagtgataca gcaaattctg atctctcttg gcttcacaat caattcaacc atgacccggg 5580
atgtagtcat acccctcctc acaaacaacg acctcttaat aaggatggca ctgttgcccg 5640
ctcctattgg ggggatgaat tatctgaata tgagcaggct gtttgtcaga aacatcggtg 5700
atccagtaac atcatcaatt gctgatctca agagaatgat tctcgcctca ctaatgcctg 5760
aagagaccct ccatcaagta atgacacaac aaccggggga ctcttcattc ctagactggg 5820
ctagcgaccc ttactcagca aatcttgtat gtgtccagag catcactaga ctcctcaaga 5880
acataactgc aaggtttgtc ctgatccata gcccaaaccc aatgttaaaa ggattattcc 5940
atgatgacag taaagaagag gacgagggac tggcggcatt cctcatggac aggcatatta 6000
tagtacctag ggcagctcat gaaatcctgg atcatagtgt cacaggggca agagagtcta 6060
ttgcaggcat gctggatacc acaaaaggcc tgattcgagc cagcatgagg aagggggggt 6120
taacatctcg agtgataacc agattgtcca attatgacta tgaacaattc agagcaggga 6180
tggtgctatt gacaggaaga aagagaaatg tcctcattga caaagagtca tgttcagtgc 6240
agctggcgag agctctaaga agccatatgt gggcgaggct agctcgagga cggcctattt 6300
acggccttga ggtccctgat gtactagaat ctatgcgagg ccaccttatt cggcgtcatg 6360
agacatgtgt catctgcgag tgtggatcag tcaactacgg atggtttttt gtcccctcgg 6420
gttgccaact ggatgatatt gacaaggaaa catcatcctt gagagtccca tatattggtt 6480
ctaccactga tgagagaaca gacatgaagc ttgccttcgt aagagcccca agtcgatcct 6540
tgcgatctgc tgttagaata gcaacagtgt actcatgggc ttacggtgat gatgatagct 6600
cttggaacga agcctggttg ttggccaggc aaagggccaa tgtgagcctg gaggagctaa 6660
gggtgatcac tcccatctca acttcgacta atttagcgca taggttgagg gatcgtagca 6720
ctcaagtgaa atactcaggt acatcccttg tccgagtggc gaggtatacc acaatctcca 6780
acgacaatct ctcatttgtc atatcagata agaaggttga tactaacttt atataccaac 6840
aaggaatgct tctagggttg ggtgttttag aaacattgtt tcgactcgag aaagataccg 6900
gatcatctaa cacggtatta catcttcacg tcgaaacaga ttgttgcgtg atcccgatga 6960
tagatcatcc caggataccc agctcccgca agctagagct gagggcagag ctatgtacca 7020
acccattgat atatgataat gcacctttaa ttgacagaga tacaacaagg ctatacaccc 7080
agagccatag gaggcacctt gtggaatttg ttacatggtc cacaccccaa ctatatcaca 7140
ttttagctaa gtccacagca ctatctatga ttgacctggt aacaaaattt gagaaggacc 7200
atatgaatga aatttcagct ctcatagggg atgacgatat caatagtttc ataactgagt 7260
ttctgctcat agagccaaga ttattcacta tctacttggg ccagtgtgcg gccatcaatt 7320
gggcatttga tgtacattat catagaccat cagggaaata tcagatgggt gagctgttgt 7380
catcgttcct ttctagaatg agcaaaggag tgtttaaggt gcttgtcaat gctctaagcc 7440
acccaaagat ctacaagaaa ttctggcatt gtggtattat agagcctatc catggtcctt 7500
cacttgatgc tcaaaacttg cacacaactg tgtgcaacat ggtttacaca tgctatatga 7560
cctacctcga cctgttgttg aatgaagagt tagaagagtt cacatttctc ttgtgtgaaa 7620
gcgacgagga tgtagtaccg gacagattcg acaacatcca ggcaaaacac ttatgtgttc 7680
tggcagattt gtactgtcaa ccagggacct gcccaccaat tcgaggtcta agaccggtag 7740
agaaatgtgc agttctaacc gaccatatca aggaagaggc taggttatct ccagcaggat 7800
cttcgtggaa cataaatcca attattgtag accattactc atgctctctg acttatctcc 7860
ggcgaggatc gatcaaacag ataagattga gagttgatcc aggattcatt ttcgacgccc 7920
tcgctgaggt aaatgtcagt cagccaaaga tcggcagcaa caacatctca aatatgagca 7980
tcaaggcttt cagaccccca cacgatgatg ttgcaaaatt gctcaaagat atcaacacaa 8040
gcaagcacaa tcttcccatt tcagggggca atctcgccaa ttatgaaatc catgctttcc 8100
gcagaatcgg gttgaactca tctgcttgct acaaagctgt tgagatatca acattaatta 8160
ggagatgcct tgagccaggg gaggacggct tgttcttggg tgagggatcg ggttctatgt 8220
tgatcactta taaggagata cttaaactaa acaagtgctt ctataatagt ggggtttccg 8280
ccaattctag atctggtcaa agggaattag caccctatcc ctccgaagtt ggccttgtcg 8340
aacacagaat gggagtaggt aatattgtca aagtgctctt taacgggagg cccgaagtca 8400
cgtgggtagg cagtgtagat tgcttcaatt tcatagttag taatatccct acctctagtg 8460
tggggtttat ccattcagat atagagacct tgcctaacaa agatactata gagaagctag 8520
aggaattggc agccatctta tcgatggctc tgctcctggg caaaatagga tcaatactgg 8580
tgattaagct tatgcctttc agcggggatt ttgttcaggg atttataagt tatgtagggt 8640
ctcattatag agaagtgaac cttgtatacc ctagatacag caacttcata tctactgaat 8700
cttatttggt tatgacagat ctcaaggcta accggctaat gaatcctgaa aagattaagc 8760
agcagataat tgaatcatct gtgaggactt cacctggact tataggtcac atcctatcca 8820
ttaagcaact aagctgcata caagcaattg tgggagacgc agttagtaga ggtgatatca 8880
atcctactct gaaaaaactt acacctatag agcaggtgct gatcaattgc gggttggcaa 8940
ttaacggacc taagctgtgc aaagaattga tccaccatga tgttgcctca gggcaagatg 9000
gattgcttaa ttctatactc atcctctaca gggagttggc aagattcaaa gacaaccaaa 9060
gaagtcaaca agggatgttc cacgcttacc ccgtattggt aagtagcagg caacgagaac 9120
ttatatctag gatcacccgc aaattttggg ggcacattct tctttactcc gggaacagaa 9180
agttgataaa taagtttatc cagaatctca agtccggcta tctgatacta gacttacacc 9240
agaatatctt cgttaagaat ctatccaagt cagagaaaca gattattatg acggggggtt 9300
tgaaacgcga gtgggttttt aaggtaacag tcaaggagac caaagaatgg tataagttag 9360
tcggatacag tgccctgatt aaggactaat tggtgtcgac ctgcagtcta gaatcgaagg 9420
gcgccaccat caccatcacc attaggtaat ctctgaaaaa aaaaaaaaaa aaaaaaaaaa 9480
aaaaaactag cataacccct tggggcctct aaacgggtct tgaggggttt tttggatcat 9540
ccgcggccgc taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 9600
ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 9660
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 9720
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccggtgcact ctcagtacaa 9780
tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 9840
cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 9900
gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcga 9945
<210> 6
<211> 31
<212> DNA
<213> MVF1 上游引物
<400> 6
ttggcgcgcc accaaacaaa gttgggtaag g 31
<210> 7
<211> 22
<212> DNA
<213> MVF1 下游引物
<400> 7
tccctgaccg cggatgcgag gt 22
<210> 8
<211> 22
<212> DNA
<213> MVF2 上游引物
<400> 8
tcgcatccgc ggtcagggac ct 22
<210> 9
<211> 25
<212> DNA
<213> MVF2 下游引物
<400> 9
tttctttccc gggggtggtg gggat 25
<210> 10
<211> 22
<212> DNA
<213> MVF3 上游引物
<400> 10
ccaccacccc cgggaaagaa ac 22
<210> 11
<211> 44
<212> DNA
<213> MVF3 下游引物
<400> 11
ccaccacccc cctaagtttt aattaactac cgcgggaaag aaac 44
<210> 12
<211> 22
<212> DNA
<213> MVF4上游引物
<400> 12
cggtagttaa ttaaaactta gg 22
<210> 13
<211> 23
<212> DNA
<213> MVF4 下游引物
<400> 13
acagcgaatt cccctttttg agg 23
<210> 14
<211> 23
<212> DNA
<213> MVF5 上游引物
<400> 14
cctcaaaaag gggaattcgc tgt 23
<210> 15
<211> 22
<212> DNA
<213> MVF5 下游引物
<400> 15
catgtgctgg gtaccctttt tg 22
<210> 16
<211> 22
<212> DNA
<213> MVF6 上游引物
<400> 16
caaaaagggt acccagcaca tg 22
<210> 17
<211> 21
<212> DNA
<213> MVF6 下游引物
<400> 17
gataatgtac atcaaatgcc c 21
<210> 18
<211> 21
<212> DNA
<213> MVF7 上游引物
<400> 18
gggcatttga tgtacattat c 21
<210> 19
<211> 37
<212> DNA
<213> MVF7 下游引物
<400> 19
ataagaatgc ggccgcacca gacaaagctg ggaatag 37
<210> 20
<211> 20
<212> DNA
<213> pT7-IRES-NotI
<400> 20
atccgcggcc gctaattcac 20
<210> 21
<211> 26
<212> DNA
<213> pT7-IRES-NheI
<400> 21
tagctagcaa gcttggcgta atcatg 26
<210> 22
<211> 113
<212> DNA
<213> 多克隆位点序列(MCS)
<400> 22
gctagctagt tggcgcgcca atccccgcgg ggatcccccc ggggggacct taattaaggc 60
cggaattccg gcggggtacc ccggcgctgt acagcgcata agaatgcggc cgc 113
<210> 23
<211> 253
<212> DNA
<213> pT7-MVF1-HamRz
<400> 23
ggcgcgccta atacgactca ctatagggcc aactttgttt ggtctgatga gtccgtgagg 60
acgaaacccg ggtcaccaaa caaagttggg taaggatagt tcaatcaatg atcatcttct 120
agtgcactta ggattcaaga tcctattatc agggacaaga gcaggattag ggatatccga 180
gatggccaca cttttaagga gcttagcatt gttcaaaaga aacaaggaca aaccacccat 240
tacatcagga tcc 253
<210> 24
<211> 307
<212> DNA
<213> pT-MVF7-HdvRz
<400> 24
tccggaaccc taatcctgcc ctaggtggtt aggcattatt tgcaatatat taaagaaaac 60
tttgaaaata cgaagtttct attcccagct ttgtctggtg gccggcatgg tcccagcctc 120
ctcgctggcg ccggctgggc aacattccga ggggaccgtc ccctcggtaa tggcgaatgg 180
gacgcggccg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 240
gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 300
cggccgc 307
<210> 25
<211> 177
<212> DNA
<213> pT7-MVF2-ATU
<400> 25
gtcgacccaa ctagcctacc ctccatcatt gttataaaaa acttaggaac caggtccaca 60
cagccgccag cccatcaacg cgtacgatgg tgagcaaggg cgaggagctg ttcaccactc 120
tcggcatgga cgagctgtac aagtaggcgc gcagcgctta gacgtctcgc gactagt 177
<210> 26
<211> 68
<212> DNA
<213> L1
<400> 26
catggccaca cttttaagga gcttagcatt gttcaaaaga aacaaggaca aaccacccat 60
tacatcag 68
<210> 27
<211> 68
<212> DNA
<213> L2
<400> 27
catggccaca cttttaagga gcttagcatt gttcaaaaga aacaaggaca aaccacccat 60
tacatcag 68
<210> 28
<211> 22
<212> DNA
<213> N-P1
<400> 28
tcaggatccg gtggagccat ca 22
<210> 29
<211> 31
<212> DNA
<213> N-P2
<400> 29
ggactagtct agtctagaag atttctgtca t 31
<210> 30
<211> 26
<212> DNA
<213> P-P1
<400> 30
aaccatggca gaagagcagg cacgcc 26
<210> 31
<211> 33
<212> DNA
<213> P-P2
<400> 31
gctctagact acttcattat tatcttcatc agc 33
<210> 32
<211> 32
<212> DNA
<213> L-P1
<400> 32
cccatggatg gactcgctat ctgtcaacca ga 32
<210> 33
<211> 20
<212> DNA
<213> L-P2
<400> 33
gtgagttagt gtcccttaag 20
Claims (11)
1.一种用于拯救麻疹病毒或重组麻疹病毒的系统，其特征在于：它包括如下成分：
1)含有麻疹病毒反基因组全长的转录质粒；
2)辅助质粒1，其含有麻疹病毒的核衣壳蛋白的编码基因；
3)辅助质粒2，其含有麻疹病毒的磷蛋白的编码基因；
4)辅助质粒3，其含有麻疹病毒的RNA聚合酶的编码基因。
2.根据权利要求1所述的系统，其特征在于：所述麻疹病毒为减毒麻疹疫苗株沪191；所述的重组麻疹病毒为表达GFP的重组麻疹病毒沪191。
3.根据权利要求1所述的系统，其特征在于：所述含有麻疹病毒反基因组全长的转录质粒为含有麻疹病毒反基因组全长的质粒pT7MVS191或pT7MVS191-ATU1-GFP；其中，所述pT7MVS191的核苷酸序列如SEQ ID NO.1所示；所述pT7MVS191-ATU1-GFP的核苷酸序列如SEQID NO.2所示。
4.根据权利要求1所述的系统，其特征在于：所述辅助质粒1的核苷酸序列如SEQ IDNO.3所示。
5.根据权利要求1所述的系统，其特征在于：所述辅助质粒2的核苷酸序列如SEQ IDNO.4所示。
6.根据权利要求1所述的系统，其特征在于：所述辅助质粒3的核苷酸序列如SEQ IDNO.5所示。
7.一种拯救麻疹病毒及重组麻疹病毒的方法，其特征在于：它是采用权利要求1-6任意一项所述的系统进行拯救，步骤如下：
(1)将权利要求中1-6任意一项系统中的质粒共转染细胞汇合度为60％～90％时的辅助细胞；
(2)37℃培养，热休克，37℃继续培养3d，得培养的上清混合物；
(3)取步骤(2)的上清混合物，感染宿主细胞，回收表达的病毒颗粒。
8.根据权利要求7所述的方法，其特征在于：步骤(1)中，所述辅助细胞为表达T7RNA聚合酶的BSR T7细胞。
9.根据权利要求7所述的方法，其特征在于：步骤(2)中，所述热休克的温度为42-44℃，优选温度为43.5℃；所述热休克时间为1-4小时，优选3小时。
10.根据权利要求7所述的方法，其特征在于：步骤(3)中，所述的宿主细胞为Veroslam细胞、人二倍体细胞或者鸡胚成纤维细胞。
11.权利要求7-10任意一项所述的方法在制备适用于治疗性和/或预防性的感染性麻疹病毒和/或重组麻疹病毒中的用途。
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Cited By (6)
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| CN110982940A (zh) * | 2019-12-30 | 2020-04-10 | 广州市疾病预防控制中心 | 基于熔解曲线的麻疹病毒、风疹病毒和腮腺炎病毒核酸检测的组合物及试剂盒 |
| CN111218475A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种拯救麻疹Schwarz/Moraten疫苗株的系统和方法 |
| CN111218474A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种副粘病毒的拯救系统和副粘病毒拯救方法 |
| CN111218473A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种拯救腮腺炎病毒的系统和方法 |
| CN111676244A (zh) * | 2020-06-05 | 2020-09-18 | 成都生物制品研究所有限责任公司 | 以麻疹病毒为载体的麻疹、风疹联合疫苗 |
| CN117904197A (zh) * | 2024-01-16 | 2024-04-19 | 伊诺维康(北京)生物科技有限公司 | 一种羊轮状病毒感染性克隆质粒和构建方法、羊轮状病毒的反向遗传病毒拯救系统及其应用 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218475A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种拯救麻疹Schwarz/Moraten疫苗株的系统和方法 |
| CN111218474A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种副粘病毒的拯救系统和副粘病毒拯救方法 |
| CN111218473A (zh) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | 一种拯救腮腺炎病毒的系统和方法 |
| CN110982940A (zh) * | 2019-12-30 | 2020-04-10 | 广州市疾病预防控制中心 | 基于熔解曲线的麻疹病毒、风疹病毒和腮腺炎病毒核酸检测的组合物及试剂盒 |
| CN111676244A (zh) * | 2020-06-05 | 2020-09-18 | 成都生物制品研究所有限责任公司 | 以麻疹病毒为载体的麻疹、风疹联合疫苗 |
| CN111676244B (zh) * | 2020-06-05 | 2024-02-23 | 成都生物制品研究所有限责任公司 | 以麻疹病毒为载体的麻疹、风疹联合疫苗 |
| CN117904197A (zh) * | 2024-01-16 | 2024-04-19 | 伊诺维康(北京)生物科技有限公司 | 一种羊轮状病毒感染性克隆质粒和构建方法、羊轮状病毒的反向遗传病毒拯救系统及其应用 |
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