CN115595314B 表达天冬氨酸脱氢酶的工程菌及发酵生产维生素b5的方法 （中国科学院微生物研究所）

表达天冬氨酸脱氢酶的工程菌及发酵生产氨酸脱氢酶基因aspDH的大肠杆菌，及其用于发于维生素B5的工业化生产，具有重要的应用价21.宿主，其特征在于，表达了天冬氨酸脱氢酶基因aspDH；所述天冬氨酸脱氢酶基因3现AspDH比AspC和AspA更有利于提高VB[0008]第一方面，本发明提供了增强天冬氨酸脱氢酶基因aspDH的表达在生产维生素B5[0009]作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl一4(Delftia4[0015](1)、在cadA基因中插入强启动子和/或强RBS，其中强启动子为PgapA，强RBS为5[0048]作为优选，所述天冬氨酸脱氢酶基因aspDH来源于戴尔福特菌Csl一4(Delftia[0068]在本发明的一些具体实施方案中，所述宿主转染或转化如权利要求4或5所述的6[0072]本发明公开了一种表达天冬氨酸脱氢酶基因aspDH的大肠杆菌，及其用于发酵生氨酸和酮戊二酸；aspA基因编码的天冬氨酸氨裂解酶，催化铵和延胡索酸产生天冬氨酸；现了催化草酰乙酸和铵合成天冬氨酸的天冬氨酸脱氢酶(Aspartatedehydrogenase，[0075]为了突破高效合成维生素B5的代谢瓶颈，发明人比较了3种提高天冬氨酸合成的途径，发现异源的天冬氨酸脱氢(aspDH基因编码)途径更适合自身的天冬氨酸转氨途径尔福特菌Csl一4(Delftiasp.Csl一4)、人苍白杆菌(Ochrobactrumanthropi)、柄杆菌(Caulobactersp.)、马氏甲烷嗜盐菌(Methanohalophilusmahii)、柴氏海洋玫瑰杆菌杆菌(Methanobrevibacterruminantiu7[0078]本发明将上述高强度的翻译起始和转录起始调控的aspDH基因整合到发酵法生产[0079]在发酵法生产VB5的大肠杆菌染色体上相同的cadA基因位点，分别整合aspA和[0080]本发明所述的发酵法生产VB5的大肠杆菌，还过表达了VB5终端合成途径上的戊酸增加一个甲基形成酮泛解酸。酮泛解酸由panE基因编码的酮泛解酸还原酶还原为泛肠杆菌K12MG1655的染色体上插入了来源于大肠杆菌BL21的ilvG+M基因，且使用trc强启的插入位点为avtA基因的编码序列，导致AvtA失活，弱化了缬氨酸的合成，从而弱化了[0082]在工程菌avtA基因上还整合了源于地衣芽孢杆菌(Bacilluslicheniformis)的8高保真聚合酶KAPAHiFiTMHotStar，PCR扩增的核苷酸序列如SEQIDNo.3所示，其中酶切上述扩增的Ptrc_panBC基因的DNA片段和pACYC184质粒。凝胶电泳回收双酶切的Ptrc_988nt_173Int为终止子序列。扩增引物P3上设计启动子PJ23119，引物P4上设计终止子酶切，同时双酶切pACYC184_Ptrc_panBC质粒。凝胶电泳回收双酶切的PJ23119_panE和[0099](如SEQIDNo.12所示，下划线所示序列为SphI酶切识别位点，斜体为启动子9[0102]应用已报道的包含pCas9和pTargetF载体的CRISPR_Cas9基因编辑系统(Jiang，[0103]使用NEB公司的基因突变试剂盒(Site_DirectedMutagenesisKit，货号E0552S)，按照试剂盒说明书设计引物P5和P6突变pTargetF载体。突变后的N20序列为[0110]P11：ATCAGCAGGACGCACTGACCCCAAGTTCACTTAAAAAGGAGATC(如SEQIDNO.20所[0116]在基因合成公司合成了来源于地衣芽孢杆菌Bacilluslicheniformis天冬氨酸子替换去除XbaI和HindIII限制性内切酶序列。在定制合成上述panD基因序列时，在每个酶切后的BCD2_panD的基因片段和线性化载体段，进一步使用T4连接酶连接这两个片段，株MG655/pCas9。挑取MG1655/pCas9菌苔于50mL含卡那霉素的LB的500mL摇瓶中，30℃,时制备感受态细胞。取2微升pTargetFavtA质粒和10微升DonorBs模板DNA，电转化至养12小时，稀释涂布LB平板，分别获得消除pCas质粒的工程菌E.coliMG1655avtA：[0121]野生型大肠杆菌K12MG1655的ilvG基因突变，其编码的乙酰乳酸合成酶没有活高了VB5的前体乙酰乳酸的合成。本发明在大肠杆菌K12MG1655的染色体上插入了来源于[0125]P20：TCCACACATTATACGAGCCGGATGAT[0126]P21：TTGACAATTAATCATCCGGCTCGTATAATGTGTGGACAAGATTCAGGACGGGGAAC(如[0127]P22：CGAAAAAAGACGCTCTAAAAGCGTCTCTTTTCTGGTATATTCCTTTTGCGCTCAG(如[0128]P23：CAGAAAAGAGACGCTTTTAGAGCGTCTTTTTTCGTTTTGGAGCTACTCACACTGCTTG(如过引物P22和P23引入终止子序列CCAGAAAAGAGACGCTTTTAGAGCGTCTTTTTTCGTTTT。使用重叠2013nt为源于E.coliBL21的ilvG+基因的编码序列，2010nt_2273nt为ilvM基因的编码序[0134]取2微升pTargetFavtA1质粒和10微升DonorilvGM模板DNA，电转化至E.Coli[0135]使用上述突变试剂盒和引物P27和P28突变pTargetF载体的N20序列，突变[0141]P32：CTCCTTAGCATGATTAAGATGGTGAATAAAAGGTTGCCTG[0143]P34：GCTAATTTCTTCGCACAGCTGGACCAAAACGAAAAAAGA[0149]取2微升pTargetFcadA质粒和10微升DonoraspDH模板DNA，电转化至E.coliMG1655avtA：panDBl_ilvG+M/p增cadA基因下游序列。通过重叠PCR连接上述4个片段，获得组合体DonoraspC(如SEQIDNo.8所示)，作为基因编辑的模板。SEQIDNo.8的1_210nt因下游序列。[0151]取2微升pTargetFcadA质粒和10微升DonoraspC模板DNA，电转化至E.coli[0152]P39：GAGATTGCTCTGGAAGGTATAGTGAATAAAAGGTTGCCTGT(如SEQIDNO.48所[0153]P40：ACAGGCAACCTTTTATTCACTATACCTTCCAGAGCAATCTC(如SEQIDNO.49所[0155]P42：CACTTTGCCAGAAATCCAGGAGCTGTGCGAAGAAATTAGC(如SEQIDNO.51所增cadA基因下游序列。通过重叠PCR连接上述4个片段，获得组合体DonoraspA(如SEQIDNo.9所示)，作为基因编辑的模板。SEQIDNo.9的1_210nt因下游序列。[0157]取2微升pTargetFcadA质粒和10微升DonoraspA模板DNA，电转化至E.coli[0158]P43：GAACCTTCTTTTTCAAGCTGCGTGAATAAAAGGTTGCCTG[0162]将上述构建的载体pACYC184_panBCE转化至上述工程菌E.coliMG1655avtA：panDBl_ilvG+M_aspDH、E.coliMG1655avtA：panDBl_ilvG+M_aspC和E.coliMG1655avtA：panDBl_ilvG+M_aspA中，分别获得工程菌E.ColiMG1655avtA：panDBl_ilvG+M_[0199]取试验菌株工程菌E.coliMG1655avtA：panDBl_ilvG+M_aspDH/pACYC184_panBCE、E.coliMG1655avtA：panDBl_ilvG+M_aspC/pACYC184_panBCE和E.coavtA：panDBl_ilvG+M_aspA/pACYC184_pa水硫酸镁2.0g/L、酵母粉5.0g/L、微量元素混合液5mL/L，余量为水。微量元素混合液：FeSO4[0204]本发明通过在发酵法生产VB5的大肠杆菌中增强三条不同的产生天冬氨酸的途[0002]<110>中国科学院微生物研究所[0003]<120>表达天冬氨酸脱氢酶的工程菌及发酵生产维生素B5的方法[0004]<130>IM2022022I[0005]<141>2022_03_07[0007]<170>SIPOSequenceListing1.0[0011]<213>人工序列(ArtificialSequence)[0013]atgtaccgtacgttaatgagcgcaaaacttcacagagcgagagtgacggaagccaatttg60[0014]aactacgtcggcagcgtgacaattgatgaagatttgctggatgctgtcggaatgatggca120[0015]aatgaaaaagtgcaaattgtgaataataataacggggcccggctggaaacgtacattatt180[0016]cccggtgaaaggggcagcggcgtcgtttgtttaaacggagctgccgcccgccttgtccag240[0017]gttggagatgtcgtcatcatcgtgtcttatgcgatgatgtctgaagaggaagcaaagacc300[0018]cataagccgaaggttgccgttttgaacgagagaaacgaaatcgaggaaatgctgggtcag360[0019]gagccagcccgtaccattctgtaa384[0023]<213>人工序列(ArtificialSequence)[0025]ccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaa60[0026]tcatgctaaggaggttttctaatg84[0030]<213>人工序列(ArtificialSequence)[0032]cgcggatccttgacaattaatcatccggctcgtataatgtgtggagcacaacatcaattt60[0033]atcaggatacgttatgaaaccgaccaccatctccttactgcagaagtacaaacaggaaaa120[0034]aaaacgtttcgcgaccatcaccgcttatgactatagcttcgccaaactctttgctgatga180[0035]agggcttaacgtcatgctggtgggcgattcgctgggcatgacggttcaggggcacgactc240[0036]caccctgccagttaccgttgccgatatcgcctaccacactgccgccgtacgtcgcggcgc300[0037]accaaactgcctgctgctggctgacctgccgtttatggcgtatgccacgccggaacaagc360[0038]cttcgaaaacgccgcaacggttatgcgtgccggtgctaacatggtcaaaattgaaggcgg420[0039]tgagtggctggtagaaaccgtacaaatgctgaccgaacgtgccgttcctgtatgtggtca480[0040]cttaggtttaacaccacagtcagtgaatattttcggtggctacaaagttcaggggcgcgg540[0041]cgatgaagcgggcgatcaactgctcagcgatgcattagccttagaagctgctggggcaca600[0042]gctgctggtgctggaatgcgtgccggttgaactggcaaaacgtattaccgaagcactggc660[0043]gatcccggttattggcattggcgcaggcaacgtcactgacgggcagatcctcgtgatgca720[0044]cgacgcctttggtattaccggcggtcacattcctaaattcgctaaaaatttcctcgccga780[0045]aacgggcgacatccgcgcggctgtgcggcagtatatggctgaagtggagtccggcgttta840[0046]tccgggcgaagaacacagtttccattaaggagtcacgttgtgttaattatcgaaaccctg900[0047]ccgctgctgcgtcagcaaattcgccgcctgcgtatggaaggcaagcgcgtggcgctggtg960[0048]cctaccatgggtaacctgcacgatggccatatgaagctggtcgacgaagccaaagcccgc1020[0049]gccgatgtggtcgtcgtcagtattttcgttaacccgatgcagttcgaccgcccggaagat1080[0050]ctggctcgttatccacggaccttgcaggaggactgcgagaagctaaacaaacgtaaagtg1140[0051]gatttagttttcgccccttcggtaaaagagatctacccgaacggtactgaaacccacact1200[0052]tacgttgacgttcctggcctttcgaccatgctggaaggtgccagccgtccgggacatttt1260[0053]cgcggcgtttcgactattgtcagcaagctgttcaacctggtccagccggacatcgcctgc1320[0054]ttcggtgaaaaagattttcagcaactggcgctgatccgcaaaatggttgccgatatgggc1380[0055]ttcgatattgagattgtcggtgtgccaattatgcgcgccaaagacggtctggcgctaagt1440[0056]tcccgtaacggttatctgacggcggaacaacgcaaaattgcgcctggtctgtacaaagtt1500[0057]ttaagttcgattgctgacaaattgcaggctggggaacgggatctcgatgaaattattacc1560[0058]attgcggggcaagaactgaatgaaaaaggcttccgcgccgatgatattcagattcgcgat1620[0059]gccgacacattgctggaagtttctgaaaccagcaaacgggcagtaattctggtagccgcc1680[0060]tggcttggcgatgctcgcctgatcgacaacaaaatggtcgagctggcgtaatacttaact1740[0061]ggcgctacggctgatggcgccagttattaatttaccccacgtcatctgtcataacacagg1800[0065]<213>人工序列(ArtificialSequence)[0067]acatgcatgcttgacagctagctcagtcctaggtataatgctagcgttgcgggtgaggag60[0068]gaacaatgaaaattaccgtattgggatgcggtgccttagggcaattatggcttacagcac120[0069]tttgcaaacagggtcatgaagttcagggctggctgcgcgtaccgcaaccttattgtagcg180[0070]tgaatctggttgagacagatggttcgatatttaacgaatcgctgaccgccaacgatcccg240[0071]attttctcgccaccagcgatctgctcctggtgacgctgaaagcatggcaggtttccgatg300[0072]ccgtcaaaagcctcgcgtccacactgcctgtaactacgccaatactgttaattcacaacg360[0073]gcatgggcaccatcgaagagttgcaaaacattcagcagccattactgatgggcaccacca420[0074]cccatgcagcccgccgcgacggcaatgtcattattcatgtggcaaacggtatcacgcata480[0075]ttggcccggcacggcaacaggacggggattacagttatctggcggatattttgcaaaccg540[0076]tgttgcctgacgttgcctggcataacaatattcgcgccgagctgtggcgcaagctggcag600[0077]tcaactgcgtgattaatccactgactgccatctggaattgcccgaacggtgaattacgtc660[0078]atcatccgcaagaaattatgcagatatgcgaagaagtcgcggcggtgatcgaacgcgaag720[0079]ggcatcatacttcagcagaagatttgcgtgattacgtgatgcaggtgattgatgccacag780[0080]cggaaaatatctcgtcgatgttgcaggatatccgcgcgctgcgccacactgaaatcgact840[0081]atatcaatggttttctcttacgccgcgcccgcgcgcatgggattgccgtaccggaaaaca900[0082]cccgcctgtttgaaatggtaaaaagaaaggagagtgaatatgagcgcatcggcactggtt960[0083]tgcctcgcccctggtagtgaagagactaattttcgaaaaaagacgctgaaaagcgtcttt1020[0084]tttcgttttgggatctaaatcgag1044[0088]<213>人工序列(ArtificialSequence)[0090]cggactggaagaagatctgtttgtctctgcgcgtccgaat[0091]ccagtttaaataccacgtcgattttgagcatctgcatatt[0092]ttgcgtctcccggccgacgaatccaacaggcaatgtgatt[0093]gcttgacgcgctggcgaatcaacacggcattccgctggtg[0094]cccgttcccgggtatcatcttcagtgaagcgcgcccgcta[0095]gtgcatgagtctcaattccgacgtctaagaaaccattatt[0096]aaataggcgtatcacgaggccctttcgtcttcacctcgag[0097]tgacatccctatcagtgatagagatactgagcacatcagc[0098]aagttcacttaaaaaggagatcaacaatgaaagcaatttt[0099]atgctaaggaggttttctaatgtaccgtacgttaatgagc[0100]agtgacggaagccaatttgaactacgtcggcagcgtgaca[0101]tgctgtcggaatgatggcaaatgaaaaagtgcaaattgtg[0102]gctggaaacgtacattattcccggtgaaaggggcagcggc[0103]tgccgcccgccttgtccaggttggagatgtcgtcatcatc[0104]tgaagaggaagcaaagacccataagccgaaggttgccgtt[0105]cgaggaaatgctgggtcaggagccagcccgtaccattctg[0106]ttggggcctctaaacgggtcttgaggggttttttgcatca[0107]agcctggcacctggcggtattggtccggcgatgatgtgtg[0108]ctgctgcgcctgtctgaaacagtcatcaaaccgttttact[0109]atcgccatcattcgccgctatttaccggaaaatcgctgcc[0110]gccattttcctctggctatggtttaaggatttgcccatta<213>人工序列(ArtificialSequence)<400>6attgaactgctgccggaagg60ggcgaagaaaccgggatgat120actgacgaagagttgctgaa180attgataacgcttatggcgt240tggaatccgaatatcgtgct300atcatgacattaacctataa360tccctatcagtgatagagat420aggacgcactgaccgggccc480cgtactgaaacatcttaatc540gcaaaacttcacagagcgag600attgatgaagatttgctgga660aataataataacggggcccg720gtcgtttgtttaaacggagc780gtgtcttatgcgatgatgtc840ttgaacgagagaaacgaaat900taaaagcctagcataacccc960ccaatatgaacggcattatc1020aaatgattaagcgtaacgat1080accagcgtgttcaggaaact1140tgattcataaaccggaagga1200cgaccaagcagctctatcag1260ggcagaaaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttttgctgaagcagggctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaacttttccggcttcccgttcgtcagtacctcgggaagccgccaaccaggataaaatgtcagccctaatcagcgttgcaggataaagcaccgctcactcttcaacagaccgatttgcaccccagcaaatgtagcgttattgttaccttccttgctacagagttcttgacaattaatcatccggctcgtataatgtgtggacaagattcaggacggggaactaactatgaatggcgcacagtgggtggtacatgcgttgcgggcacagggtgtgaacacc420gttttcggttatccgggtggcgcaattatgccggtttacgatgcattgtatgacggcggc480gtggagcacttgctatgccgacatgagcagggtgcggcaatggcggctatcggttatgct[0126]cgtgctaccggcaaaactggcgtatgtatcgccacgtctggtccgggcgcaaccaacctg600[0127]ataaccgggcttgcggacgcactgttagattccatccctgttgttgccatcaccggtcaa660[0128]gtgtccgcaccgtttatcggcactgacgcatttcaggaagtggatgtcctgggattgtcg720[0129]ttagcctgtaccaagcacagctttctggtgcagtcgctggaagagttgccgcgcatcatg780[0130]gctgaagcattcgacgttgcctgctcaggtcgtcctggtccggttctggtcgatatccca840[0131]aaagatatccagttagccagcggtgacctggaaccgtggttcaccaccgttgaaaacgaa900[0132]gtgactttcccacatgccgaagttgagcaagcgcgccagatgctggcaaaagcgcaaaaa960[0133]ccgatgctgtacgttggcggtggcgtgggtatggcgcaggcagttccggctttgcgtgaa1020[0134]tttctcgctgccacaaaaatgcctgccacctgtacgctgaaagggctgggcgcagtagaa1080[0135]gcagattatccgtactatctgggcatgctgggaatgcatggcaccaaagcggcgaacttc1140[0136]gcggtgcaggagtgcgacttgctgatcgccgtgggtgcacgttttgatgaccgggtgacc1200[0137]ggcaaactgaacaccttcgcaccacacgccagtgttatccatatggatatcgacccggca1260[0138]gaaatgaacaagctgcgtcaggcacatgtggcattacaaggtgatttaaatgctctgtta1320[0139]ccagcattacagcagccgttaaatatcaatgactggcagctacactgcgcgcagctgcgt1380[0140]gatgaacatgcctggcgttacgaccatcccggtgacgctatctacgcgccgttgttgtta1440[0141]aaacaactgtcagatcgtaaacctgcggattgcgtcgtgaccacagatgtggggcagcac1500[0142]cagatgtgggctgcgcagcacatcgcccacactcgcccggaaaatttcatcacctccagc1560[0143]ggcttaggcaccatgggttttggtttaccggcggcggttggcgcgcaagtcgcgcgacca1620[0144]aacgataccgtcgtctgtatctccggtgacggctctttcatgatgaatgtgcaagagctg1680[0145]ggcaccgtaaaacgcaagcagttaccgttgaaaatcgtcttactcgataaccaacggtta1740[0146]gggatggttcgacaatggcagcaactgtttttccaggaacgatatagcgaaaccaccctt1800[0147]accgataaccccgatttcctcatgttagccagcgccttcggcatccctggccaacacatc1860[0148]acccgtaaagaccaggttgaagcggcactcgacaccatgctgaacagtgatgggccatac1920[0149]ctgcttcatgtctcaatcgacgaacttgagaacgtctggccgctggtgccgcctggtgcc1980[0150]agtaattcagaaatgttggagaaattatcatgatgcaacatcaggtcaatgtatcggctc2040[0151]gcttcaatccagaaaccttagaacgtgttttacgcgtggtgcgtcatcgtggtttccacg2100[0152]tctgctcaatgaatatggccgccgccagcgatgcacaaaatataaatatcgaattgaccg2160[0153]ttgccagcccacggtcggtcgacttactgtttagtcagttaaataaactggtggacgtcg2220[0154]cacacgttgccatctgccagagcacaaccacatcacaacaaatccgcgcctgagcgcaaa2280[0155]aggaatataccagaaaagagacgcttttagagcgtcttttttcgttttggagctactcac2340[0156]actgcttgccggaatgctgcgcgagaagttgggttgggatatcgaaccacagaatattgc2400[0157]actaacaaacggcagccagagcgcgtttttctacttatttaacctgtttgccggacgccg2460[0158]tgccgatggtcgggtcaaaaaagtgctgttcccgcttgcaccggaatacattggctatgc2520[0159]tgacgccggactggaagaagatctgtttgtctctgcgcgtccgaatattgaactgctgcc2580[0160]ggaaggccagtttaaataccacgtcgattttgagcatctgcatattggc2629[0164]<213>人工序列(ArtificialSequence)[0166]gttgcgtgttctgcttcatcgcgctgatgggcgcaagctccttcgagctggcaggtacct60[0167]tcatcgtcagcctgattatcctgatgttctacgctcgcaaaatgcacgagcgccagagcc120actcaatggataaccacaccgcgtctaacgcacattaattctcctttcattttgtcccatgtgttgggagcaaaacattagtcctaaacttgatctcgacgaaatggctgcacctaaatcatcgtaattgccctttaaaattcggggcgccgaccccatggagtgaggcgagtcagtcgcgtaatgcttaggcacaggatcacgattccgcttgacgctgcgtaaggtttttgtaattttcatcttaatcatgctaaggaggttttctaatgaatattgcttggcgccagcgtgctcgaactgctcaagggccatgccgcttgtgcccgaagtgacggacgccgtgcgcgccaccctggcgcgcactgcctgcgctgacgactgaagaccggcccgacctataccgccatcgaagagcatgtgctgcccgccctgcggcgcctccatcggcgcactcagcgcccccggcatggccgaggcccggaggcacccaggtgcaattgctgtcgggcgccatcggcagcccgcatcggcggcctggacgaagtggtctacaccggggaccggcacgcccgcagaacagcgctgcgacctcgccagtcttcgaaggcagcgcacgcgaggccgcccagctctacccccaccctgtcgctggccggcatgggcctggaccgcaccaccggccgtggacgaaaacgtgcaccatgtggccgcgcgcggtgaccatgcgcggcaagccgctggaggccaaccccaagacgcgtggtgcgcgccgtgctcaaccaggccacggccatcgcgaaaaaagacgctgaaaagcgtcttttttcgttttggtccaaaatgaacgagaacctgccgttgtacgcgttcgctaataagcctgaatgacctgcgtttacagattagcttctttgaatgatattgctaataagatcaagcagaccactgacga1535[0195]<213>人工序列(ArtificialSequence)aaaagtatttacaccaactggtgatgaaaatggtctcaagtgatttgtcgacaggcaacctgtgattggcggtgcaggtgccggcatgcgtatcgtcgaacggcattcctcgttcaggcccggcgtggatccgcaagccgcctcaaggaacaagaacgccggtgcgcctgcgccttcggtctcggccctccatctaagccagctgtgcgacgtattccacatgcgctgggtccgaggctcgcaaaatttttcacatttttcccaaaggaacaatgattgattttattcactgcggtgcgaggctgggtgcccccaggcgctgcgcaggccgccgtcgtgggcggccgagggcgctggccgcccctggcctgccttctgcaaacgtggccgtacgccgacctccatggaataccgtctacagcaccttttcagaaattagctctcgatgtatgctgctgaa240420480600660840900960gttgcgtgttctgcttcatcgcgctgatgggcgcaagctccttcgagctggcaggtacct60tcatcgtcagcctgattatcctgatgttctacgctcgcaaaatgcacgagcgccagagccactcaatggataaccacaccgcgtctaacgcacattaattaaaagtattttccgaggctcctcctttcattttgtcccatgtgttgggagcaaaacattaacaccaactggcaaaattttgtcctaaacttgatctcgacgaaatggctgcacctaaatcgtgatgaaaatcacatttttatcgtaattgccctttaaaattcggggcgccgaccccatgtggtctcaagcccaaaggaagagtgaggcgagtcagtcgcgtaatgcttaggcacaggattgatttgtcgcaatgattga420cacgattccgcttgacgctgcgtaaggtttttgtaattttacaggcaaccttttattcac480tataccttccagagcaatctcacgtcttgcaaaaacagcctgcgttttcatcagtaatagttggaattttgtaaatctcccgttaccctgatagcggacttcccttctgtaaccataatg600gaacctcgtcatgtttgagaacattaccgccgctcctgccgacccgattctgggcctggc660cgatctgtttcgtgccgatgaacgtcccggcaaaattaacctcgggattggtgtctataaagatgagacgggcaaaaccccggtactgaccagcgtgaaaaaggctgaacagtatctgct[0210]cgaaaatgaaaccaccaaaaattacctcggcattgacggcatccctgaatttggtcgctg840[0211]cactcaggaactgctgtttggtaaaggtagcgccctgatcaatgacaaacgtgctcgcac900[0212]ggcacagactccggggggcactggcgcactacgcgtggctgccgatttcctggcaaaaaa960[0213]taccagcgttaagcgtgtgtgggtgagcaacccaagctggccgaaccataagagcgtctt1020[0214]taactctgcaggtctggaagttcgtgaatacgcttattatgatgcggaaaatcacactct1080[0215]tgacttcgatgcactgattaacagcctgaatgaagctcaggctggcgacgtagtgctgtt1140[0216]ccatggctgctgccataacccaaccggtatcgaccctacgctggaacaatggcaaacact1200[0217]ggcacaactctccgttgagaaaggctggttaccgctgtttgacttcgcttaccagggttt1260[0218]tgcccgtggtctggaagaagatgctgaaggactgcgcgctttcgcggctatgcataaaga1320[0219]gctgattgttgccagttcctactctaaaaactttggcctgtacaacgagcgtgttggcgc1380[0220]ttgtactctggttgctgccgacagtgaaaccgttgatcgcgcattcagccaaatgaaagc1440[0221]ggcgattcgcgctaactactctaacccaccagcacacggcgcttctgttgttgccaccat1500[0222]cctgagcaacgatgcgttacgtgcgatttgggaacaagagctgactgatatgcgccagcg1560[0223]tattcagcgtatgcgtcagttgttcgtcaatacgctgcaggaaaaaggcgcaaaccgcga1620[0224]cttcagctttatcatcaaacagaacggcatgttctccttcagtggcctgacaaaagaaca1680[0225]agtgctgcgtctgcgcgaagagtttggcgtatatgcggttgcttctggtcgcgtaaatgt1740[0226]ggccgggatgacaccagataacatggctccgctgtgcgaagcgattgtggcagtgctgta1800[0227]agcattaaaaacaatgaagcccgctgaaaagcgggctgagactgatgacaaacgcaacat1860[0228]tgcctgatgcgctacgcttatcaggcctacgcgtcccctgcaatattttgaatttgcacg1920[0229]attttgtaggccggataaggcgctcgtgccgcatccggcataaacaaagcgcactttgcc1980[0230]agaaatccaggagctgtgcgaagaaattagcaaaatgaacgagaacctgccgttgtacgc2040[0231]gttcgctaatacgtattccactctcgatgtaagcctgaatgacctgcgtttacagattag2100[0232]cttctttgaatatgcgctgggtgctgctgaagatattgctaataagatcaagcagaccac2160[0237]<213>人工序列(ArtificialSequence)[0239]gttgcgtgttctgcttcatcgcgctgatgggcgcaagctccttcgagctggcaggtacct60[0240]tcatcgtcagcctgattatcctgatgttctacgctcgcaaaatgcacgagcgccagagcc120[0241]actcaatggataaccacaccgcgtctaacgcacattaattaaaagtattttccgaggctc180[0242]ctcctttcattttgtcccatgtgttgggagcaaaacattaacaccaactggcaaaatttt240[0243]gtcctaaacttgatctcgacgaaatggctgcacctaaatcgtgatgaaaatcacattttt300[0244]atcgtaattgccctttaaaattcggggcgccgaccccatgtggtctcaagcccaaaggaa360[0245]gagtgaggcgagtcagtcgcgtaatgcttaggcacaggattgatttgtcgcaatgattga420[0246]cacgattccgcttgacgctgcgtaaggtttttgtaattttacaggcaaccttttattcac480[0247]gcagcttgaaaaagaaggttcacatgtcaaacaacattcgtatcgaagaagatctgttgg540[0248]gtaccagggaagttccagctgatgcctactatggtgttcacactctgagagcgattgaaa600[0249]acttctatatcagcaacaacaaaatcagtgatattcctgaatttgttcgcggtatggtaa660[0250]tggttaaaaaagccgcagctatggcaaacaaagagctgcaaaccattcctaaaagtgtag720[0251]cgaatgccatcattgccgcatgtgatgaagtcctgaacaacggaaaatgcatggatcagt780[0252]tcccggtagacgtctaccagggcggcgcaggtacttccgtaaacatgaacaccaacgaag840[0253]tgctggccaatatcggtctggaactgatgggtcaccaaaaaggtgaatatcagtacctga900[0254]acccgaacgaccatgttaacaaatgtcagtccactaacgacgcctacccgaccggtttcc960[0255]gtatcgcagtttactcttccctgattaagctggtagatgcgattaaccaactgcgtgaag1020[0256]gctttgaacgtaaagctgtcgaattccaggacatcctgaaaatgggtcgtacccagctgc1080[0257]aggacgcagtaccgatgaccctcggtcaggaattccgcgctttcagcatcctgctgaaag1140[0258]aagaagtgaaaaacatccaacgtaccgctgaactgctgctggaagttaaccttggtgcaa1200[0259]cagcaatcggtactggtctgaacacgccgaaagagtactctccgctggcagtgaaaaaac1260[0260]tggctgaagttactggcttcccatgcgtaccggctgaagacctgatcgaagcgacctctg1320[0261]actgcggcgcttatgttatggttcacggcgcgctgaaacgcctggctgtgaagatgtcca1380[0262]aaatctgtaacgacctgcgcttgctctcttcaggcccacgtgccggcctgaacgagatca1440[0263]acctgccggaactgcaggcgggctcttccatcatgccagctaaagtaaacccggttgttc1500[0264]cggaagtggttaaccaggtatgcttcaaagtcatcggtaacgacaccactgttaccatgg1560[0265]cagcagaagcaggtcagctgcagttgaacgttatggagccggtcattggccaggccatgt1620[0266]tcgaatccgttcacattctgaccaacgcttgctacaacctgctggaaaaatgcattaacg1680[0267]gcatcactgctaacaaagaagtgtgcgaaggttacgtttacaactctatcggtatcgtta1740[0268]cttacctgaacccgttcatcggtcaccacaacggtgacatcgtgggtaaaatctgtgccg1800[0269]aaaccggtaagagtgtacgtgaagtcgttctggaacgcggtctgttgactgaagcggaac1860[0270]ttgacgatattttctccgtacagaatctgatgcacccggcttacaaagcaaaacgctata1920[0271]ctgatgaaagcgaacagtaatcgtacagggtagtacaaataaaaaaggcacgtcagatga1980[0272]cgtgccttttttcttgtgagcagagctgtgcgaagaaattagcaaaatgaacgagaacct2040[0273]gccgttgtacgcgttcgctaatacgtattccactctcgatgtaagcctgaatgacctgcg2100[0274]tttacagattagcttctttgaatatgcgctgggtgctgctgaagatattgctaataagat2160[0275]caagcagaccactgacga2178[0279]<213>人工序列(ArtificialSequence)[0281]cgcggatcccaattaatcatccggctcgtataatgtgtgg[0286]<213>人工序列(ArtificialSequence)[0288]acatgcatgccctgtgttatgacagatgac30[0292]<213>人工序列(ArtificialSequence)agcacaacatcaatttatca60[0294]acatgcatgcttgacagctagctcagtcctaggtataatgctagcgttgcgggtgaggag60[0299]<213>人工序列(ArtificialSequence)[0301]ctcgatttagatcccaaaacgaaaaaagacgcgcttttcagcgtcttttttcgaaaatta60[0302]gtctcttcactaccagggatgactatcgag90[0306]<213>人工序列(ArtificialSequence)[0308]tgggtctaccgttttagagctagaaatagc30[0312]<213>人工序列(ArtificialSequence)[0314]gcttggaaaggactagtattatacctagg29[0318]<213>人工序列(ArtificialSequence)[0320]cggactggaagaagatctg19[0324]<213>人工序列(ArtificialSequence)[0326]tttcttagacgtcggaattgagactcatgcacagcacga39[0330]<213>人工序列(ArtificialSequence)[0332]tcgtgctgtgcatgagtctcaattccgacgtctaagaaac40[0336]<213>人工序列(ArtificialSequence)[0338]gatctcctttttaagtgaacttggggtcagtgcgtcctgctgat44[0342]<213>人工序列(ArtificialSequence)[0344]atcagcaggacgcactgaccccaagttcacttaaaaaggagatc44[0348]<213>人工序列(ArtificialSequence)[0350]tgccgttcatattggtgatgcaaaaaacccctcaagacc39[0354]<213>人工序列(ArtificialSequence)[0356]ggtcttgaggggttttttgcatcaccaatatgaacggca39[0360]<213>人工序列(ArtificialSequence)[0362]gctgatagagctgcttggt19[0366]<213>人工序列(ArtificialSequence)[0368]ggagctactcacactgcttg20[0372]<213>人工序列(ArtificialSequence)[0374]cgcatacattgatgcgtatg20[0378]<213>人工序列(ArtificialSequence)[0380]acggtccacagttttagagctagaaatagc30[0384]<213>人工序列(ArtificialSequence)[0386]cgtagttacagactagtattatacctagg29[0390]<213>人工序列(ArtificialSequence)[0392]ggcagaaaatcagccagttc20[0396]<213>人工序列(ArtificialSequence)[0398]tccacacattatacgagccggatgattaattgtcaagaactctgtagcaaggaagg56[0402]<213>人工序列(ArtificialSequence)[0404]ttgacaattaatcatccggctcgtataatgtgtggacaagattcaggacggggaac56[0408]<213>人工序列(ArtificialSequence)[0410]cgaaaaaagacgctctaaaagcgtctcttttctggtatattccttttgcgctcag55[0414]<213>人工序列(ArtificialSequence)[0416]cagaaaagagacgcttttagagcgtcttttttcgttttggagctactcacactgcttg58[0420]<213>人工序列(ArtificialSequence)[0422]gccaatatgcagatgctcatgagcatctgcatattggc38[0426]<213>人工序列(ArtificialSequence)[0428]cacgttcggatatgaactg19[0432]<213>人工序列(ArtificialSequence)[0434]cgtcaagcttcagcaactc19[0438]<213>人工序列(ArtificialSequence)[0440]tcatatctccgttttagagctagaaatagc30[0444]<213>人工序列(ArtificialSequence)[0446]ctatgaacgtgactagtattatacctagg29[0450]<213>人工序列(ArtificialSequence)[0452]gttgcgtgttctgcttcatc20[0456]<213>人工序列(ArtificialSequence)[0458]ccagttggtgttaatgttttgctcccaacacatgggaca39[0462]<213>人工序列(ArtificialSequence)[0464]tgtcccatgtgttgggagcaaaacattaacaccaactgg39[0468