调节csr系统以生产赖氨酸和赖氨酸衍生产物
阅读说明：本技术 调节csr系统以生产赖氨酸和赖氨酸衍生产物 (Modulation of CSR System to produce lysine and lysine derived products ) 是由 周豪宏 陈玲 雷云凤 刘修才 于 2018-01-09 设计创作，主要内容包括：本公开涉及遗传修饰的微生物,其过表达生物膜扩散相关多肽以增强微生物的赖氨酸和赖氨酸衍生物的生产,产生这种微生物的方法以及使用所述遗传修饰的微生物生产赖氨酸和赖氨酸衍生物的方法。(The present disclosure relates to genetically modified microorganisms that overexpress biofilm diffusion-associated polypeptides to enhance production of lysine and lysine derivatives by the microorganisms, methods of producing such microorganisms, and methods of producing lysine and lysine derivatives using the genetically modified microorganisms.)
具体实施方式
得以更详细地描述。提供以下实施例只是示例性目的，并不以任何方式限制本发明。本领域技术人员将容易地认识到各种非关键参数，可以对其进行改变或修改以产生实质上相同的结果。
实施例
提供如下实施例只是示例，并非限制请求保护的发明。
实施例1:编码CadA的质粒载体的构建
使用PCR引物cadA-F和cadA-R从大肠杆菌MG1655 K12基因组DNA扩增含有编码赖氨酸脱羧酶CadA(SEQ ID NO:2)的野生型大肠杆菌cadA(SEQ ID NO:1)的质粒载体，用限制酶SacI和BamHI消化，并连接进pSTV28以产生质粒pCIB39。使用PCR引物cadA-F2和cadA-R2优化cadA基因上游的5'序列以产生pCIB40。使用SacI-F和SacI-R引物将SacI限制性位点加回pCIB40以产生pCIB41。
实施例2:表达编码CsrA或CsrD的基因的质粒载体的构建
使用PCR引物csrA-F和csrA-R从大肠杆菌MG1655 K12基因组DNA扩增编码碳储存调节剂CsrA(SEQ ID NO:4)的大肠杆菌基因csrA(SEQ ID NO:3)，用限制酶SacI和BamHI消化及连接进也用SacI和BamHI消化的pCIB41质粒载体中以产生pCIB49。相似地，使用PCR引物csrD-F和csrD-R从大肠杆菌MG1655 K12基因组DNA扩增编码CsrD(SEQ ID NO:6)的csrD(SEQ ID NO:5)，使用sewing PCR及引物rmvBamHI-F和rmvBamHI-R除去BamHI限制位点，用限制酶SacI和BamHI消化，并连接进也用SacI和BamHI消化的pCIB41质粒载体中以产生pCIB50。
实施例3：共表达含有来自赖氨酸生物合成途径的三种蛋白质(LysC，DapA，LysA) 的合成操纵子I的质粒载体的构建
来自大肠杆菌的三个基因lysC、dapA和lysA编码参与大肠杆菌赖氨酸生物合成途径的蛋白质：天冬氨酸激酶(LysC或AKIII，由lysC编码)，二氢吡啶二羧酸合酶(DapA或DHDPS，由dapA编码)和二氨基庚二酸脱羧酶(LysA，由lysA编码)。将这三个基因克隆到质粒载体中并在大肠杆菌中过表达这三种蛋白质LysC(SEQ ID NO:7)，DapA(SEQ ID NO:8)和LysA(SEQ ID NO:9)。使用引物lysC-F和lysC-R从大肠杆菌MG1655 K12基因组DNA扩增基因lysC，使用SacI和BamHI消化扩增的片段并连接到pUC18中产生pCIB7。使用引物dapA-F和dapA-R从大肠杆菌MG1655 K12基因组DNA扩增基因dapA，使用BamHI和XbaI消化扩增的片段并连接到pCIB7中产生pCIB8。使用引物lysA-F和lysA-R从大肠杆菌MG1655 K12基因组DNA扩增基因lysA，使用XbaI和SalI消化扩增的片段并连接到pCIB8中产生pCIB9。使用引物lysC-F和lysA-R从pCIB9扩增所述三基因操纵子。使用SacI和SalI消化扩增的产物，并将消化的片段连接到pCIB10中产生pCIB32。
实施例4：共表达不同天冬氨酸激酶的质粒载体的构建。表达不同天冬氨酸激酶以 增加赖氨酸生产。
选择了两对突变，其使大肠杆菌LysC对赖氨酸具有增加的反馈抗性。使用引物318-F、318-R、323-F、323-R构建编码第一突变体LysC-1(M318I，G323D)(SEQ ID NO:10)的基因。将编码LysC-1的基因(M318I，G323D)克隆到pCIB32中并置换野生型大肠杆菌天冬氨酸激酶LysC以产生质粒pCIB43。以前曾提出但未证明来自链霉菌菌株的能产生聚赖氨酸的天冬氨酸激酶与大肠杆菌天冬氨酸激酶相比，对赖氨酸更具反馈抗性。如此，对来自变铅青链霉菌(Streptomyces lividans)的天冬氨酸激酶基因进行密码子优化，合成并克隆进pCIB32中代替野生型lysC以便使用引物SlysC-F和SlysC-R产生质粒pCIB55。表达的所得天冬氨酸激酶蛋白被称为S-LysC(SEQ ID NO:11)。
实施例5：共表达含有来自赖氨酸生物合成途径的三种蛋白质(Asd，DapB，DapD，AspC)的合成操纵子II的质粒载体的构建
接下来，增强参与大肠杆菌的赖氨酸生物合成途径的四个另外基因asd、dapB、dapD和aspC的表达。这些基因编码以下酶：天冬氨酸半醛脱氢酶(Asd(SEQ ID NO:12)，由asd编码)，二氢吡啶甲酸还原酶(DapB或DHDPR(SEQ ID NO:13)，由dapB编码)，四氢吡啶二羧酸琥珀酰酶(DapD(SEQ ID NO:14，由dapD编码)及天冬氨酸转氨酶(AspC(SEQ ID NO:15)，由aspC编码)。使用引物asd-F和asd-R从大肠杆菌MG1655 K12基因组DNA扩增基因asd，使用SacI和BamHI消化扩增的片段并连接到pUC18中产生pCIB12。使用引物dapB-F和dapB-R从大肠杆菌MG1655 K12基因组DNA扩增基因dapB，使用BamHI和XbaI消化扩增的片段并连接到pCIB12中产生pCIB13。使用引物dapD-F和dapD-R从大肠杆菌MG1655 K12基因组DNA扩增基因dapD，使用XbaI和SalI消化扩增的片段并连接到pCIB13中产生pCIB14。类似地，使用引物aspC-F和aspc-R从大肠杆菌MG1655 K12基因组DNA扩增基因aspC，使用XbaI和SalI消化扩增的片段并连接到pCIB13中产生pCIB31。
实施例6：共表达含有来自赖氨酸生物合成途径的蛋白质的合成操纵子I和II的质 粒载体的构建
使用引物lysC-rbs2-F和lysC-rbs2-R进一步优化合成操纵子I以修饰pCIB43并产生质粒pCIB378。使用引物asd-rbs2-F和asd-rbs2-R进一步优化合成操纵子II以修饰pCIB31并产生质粒pCIB380。使用引物SacI-F2、SacI-R2、ApaI-F和ApaI-R进一步修饰pCIB380以将ApaI和SacI的限制酶位点加入pCIB380中产生质粒pCIB393。将由基因lysC、dapA、lysA、asd、dapB和aspC组成的两个合成操纵子-合成操纵子I和合成操纵子II-组合进一个载体中。使用引物LAL2-SacI-F和LAL2-ApaI-R从pCIB378扩增由基因lysC、dapA和lysA组成的操纵子，用限制酶SacI和ApaI消化并连接到pCIB393中产生质粒pCIB394。
实施例7：使用大气压和室温等离子体突变大肠杆菌。
使用大气压和室温等离子体方法(ARTP)诱变大肠杆菌MG1655 K12(Zhang etal.,Appl.Microbiol.Biotechnol.98:5387-5396,2014)。ARTP II-S设备购自WuXiTMAXTREE Biotechnoogy Co.，Ltd.。将过夜培养的细胞接种到新鲜LB培养基中，生长至OD值为0.6，之后将细胞用等离子体处理50、70和90秒。洗涤三个样品中的每一个并用CGXII培养基稀释(Keilhauer et al.,J.Bacteriol.175:5595-5603,1993)。
实施例8：选择示出对S-(β-氨乙基)l-半胱氨酸抗性的突变的大肠杆菌
将每个诱变的大肠杆菌MG1655 K12样品铺板在含有0、100、200、400、600、1000或2000mg/L的S-(β-氨基乙基)l-半胱氨酸(AEC)的LB琼脂平板上。将细胞在37℃生长2天，然后计数每个平板上生长的菌落数。经过多轮ARTP诱变及在含有AEC的LB琼脂平板上筛选后，有可能获得能在含有2000mg/L AEC的平板上生长的菌落。
实施例9：使用ARTP产生的突变大肠杆菌的赖氨酸生产
测定能在2000mg/L AEC上生长的菌落的赖氨酸生产能力。将每个菌落在37℃在含有4％葡萄糖、0.1％KH2PO4、0.1％MgSO4、1.6％(NH4)2SO4、0.001％FeSO4、0.001％MnSO4、0.2％酵母提取物、0.05％L-甲硫氨酸、0.01％L-苏氨酸和0.005％L-异亮氨酸的3mL培养基中生长过夜。第二天，将每个培养物接种到含有30g/L葡萄糖和0.7％Ca(HCO3)2的100mL新鲜培养基中。将培养物在37℃生长72小时，此时测定每个培养物中的赖氨酸浓度(表1)。
表1：使用ARTP突变的大肠杆菌菌株的赖氨酸生产
如表1所示，选自含有AEC的LB平板的所有突变体均表现出过量生产赖氨酸的能力。具体地，突变体M3、M11和M15能够生产>2.0g/L。
实施例10：过表达合成操纵子I和II及CsrA或CsrD的突变大肠杆菌的赖氨酸生产
使用以下质粒组合之一转化大肠杆菌突变体M11：pCIB394和pSTV28，pCIB394和pCIB49，或者pCIB394和pCIB50。将每个转化的三个单菌落在37℃在含有4％葡萄糖、0.1％KH2PO4、0.1％MgSO4、1.6％(NH4)2SO4、0.001％FeSO4、0.001％MnSO4、0.2％酵母提取物、0.05％L-甲硫氨酸、0.01％L-苏氨酸、0.005％L-异亮氨酸、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的3mL培养基中生长过夜。第二天，将每个培养物接种到含有30g/L葡萄糖、0.7％Ca(HCO3)2、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的100mL新鲜培养基中。将培养物在37℃生长72小时，此时确定每个培养物中的赖氨酸浓度(表2)。
表2：含有合成操纵子I和II及CsrA或CsrD的突变大肠杆菌的赖氨酸生产
如表2所示，CsrA或CsrD的过量产生没有导致赖氨酸生产的增加。令人惊讶地，csrA表达的增加导致赖氨酸生产减少。
实施例11：表达sRNAs CsrB或CsrC的质粒载体的构建
使用PCR引物csrB-F和csrB-R从大肠杆菌MG1655 K12基因组DNA扩增编码CsrB的大肠杆菌csrB(SEQ ID NO:16)，用限制酶SacI和BamHI消化并连接到也用SacI和BamHI消化的pCIB41质粒载体中产生pCIB51。类似地，使用PCR引物csrC-F和csrC-R从大肠杆菌MG1655K12基因组DNA扩增csrC(SEQ ID NO:17)，用限制酶SacI和BamHI消化并连接到也用SacI和BamHI消化的pCIB41质粒载体中产生pCIB52。
实施例12：过表达合成操纵子I和II以及CsrB或CsrC的大肠杆菌的赖氨酸生产
用以下质粒组合之一转化大肠杆菌突变体M11：pCIB394和pSTV28，pCIB394和pCIB51，或者pCIB394和pCIB52。将每个转化的三个单菌落在37℃在含有4％葡萄糖、0.1％KH2PO4、0.1％MgSO4、1.6％(NH4)2SO4、0.001％FeSO4、0.001％MnSO4、0.2％酵母提取物、0.05％L-甲硫氨酸、0.01％L-苏氨酸、0.005％L-异亮氨酸、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的3mL培养基中生长过夜。第二天，将每个培养物接种到含有30g/L葡萄糖、0.7％Ca(HCO3)2、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的100mL新鲜培养基中。将培养物在37℃生长72小时，此时确定每个培养物中的赖氨酸浓度(表3)。
表3：含有合成操纵子I和II以及CsrB或CsrC的突变大肠杆菌M11的赖氨酸生产。
如表3所示，与对照相比，CsrB或CsrC的过量产生导致赖氨酸生产增加(7.5或7.6g/L比6.8g/L)。
实施例13：编码赖氨酸脱羧酶和CsrB或CsrC的质粒载体的构建
使用引物csrB-F2和csrB-R2在pCIB51上扩增csrB sRNA，使用限制酶BamHI和SphI消化扩增的片段并连接到pCIB41中形成质粒pCIB104。类似地，使用引物csrC-F2和csrC-R2扩增csrC sRNA，使用限制酶BamHI和SphI消化扩增的片段并连接到pCIB41中形成质粒pCIB105。
实施例14：共同过表达编码赖氨酸脱羧酶、CsrB或CsrC的基因以及赖氨酸合成操 纵子I和II的大肠杆菌的赖氨酸生产
用以下质粒组合之一转化大肠杆菌MG1655 K12：pCIB394和pSTV28，pCIB394和pCIB41，pCIB394和pCIB104，或pCIB394和pCIB105。将来自每个转化的三个单菌落在37℃在含有4％葡萄糖、0.1％KH2PO4、0.1％MgSO4、1.6％(NH4)2SO4、0.001％FeSO4、0.001％MnSO4、0.2％酵母提取物、0.05％L-甲硫氨酸、0.01％L-苏氨酸、0.005％L-异亮氨酸、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的3mL培养基中过夜生长。第二天，将每个培养物接种到含有30g/L葡萄糖、0.7％Ca(HCO3)2、氨苄青霉素(100μg/mL)和氯霉素(20μg/mL)的100mL新鲜培养基中。将培养物在37℃生长72小时，此时测定每个培养物中的赖氨酸和尸胺浓度(表4)。
表4：含有赖氨酸合成操纵子I和II以及过量产生赖氨酸脱羧酶和亚胺/烯胺脱氨酶的大肠杆菌菌株的赖氨酸和尸胺生产。
如表4所示，CadA的过量产生导致尸胺生产。此外，sRNA CsrB或CsrC的过量产生进一步增加尸胺生产，对于CsrB和CsrC，从3.2g/L至3.8g/L。
尽管为了清楚理解的目的，已经通过示例和实施例的方式详细地描述了本发明，但是本领域技术人员将理解，可以在所附权利要求的范围内进行一定改变和修改。本文引用的所有出版物、专利、登录号和专利申请均以引用方式并入本文以公开和描述与所引用出版物有关的方法和/或材料。在本申请与本文提供的参考文献之间存在冲突的情况下，以本申请为准。
实施例中使用的质粒表
实施例中使用的引物序列表
示例序列
SEQ ID NO:1大肠杆菌cadA核酸序列
ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA
SEQ ID NO:2 CadA多肽序列
MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA DGRYTVKVLKEESKK
SEQ ID NO:3大肠杆菌csrA核酸序列
ATGCTGATTCTGACTCGTCGAGTTGGTGAGACCCTCATGATTGGGGATGAGGTCACCGTGACAGTTTTAGGGGTAAAGGGCAACCAGGTACGTATTGGCGTAAATGCCCCGAAGGAAGTTTCTGTTCACCGTGAAGAGATCTACCAGCGTATCCAGGCTGAAAAATCCCAGCAGTCCAGTTACTAA
SEQ ID NO:4 CsrA多肽序列
MLILTRRVGETLMIGDEVTVTVLGVKGNQVRIGVNAPKEVSVHREEIYQRIQAEKSQQSSY
SEQ ID NO:5大肠杆菌csrD核酸序列
ATGAGATTAACGACGAAATTTTCGGCCTTTGTTACGCTGCTCACCGGGTTAACAATTTTTGTGACTTTGCTGGGCTGTTCGCTAAGTTTCTACAACGCCATTCAGTATAAGTTTAGTCATCGCGTTCAGGCGGTGGCGACGGCGATTGATACCCACCTTGTGTCGAATGACTTCAGCGTATTAAGGCCACAAATTACCGAATTAATGATGTCGGCAGATATCGTTCGTGTAGACCTGCTCCATGGTGATAAACAGGTTTATACCCTGGCCAGAAATGGTAGTTATCGTCCAGTTGGCTCCAGCGATCTGTTTCGCGAACTGAGCGTTCCGTTGATAAAGCATCCGGGGATGTCGTTGCGTCTGGTTTATCAGGACCCGATGGGCAACTATTTCCATTCGTTGATGACCACCGCGCCGCTCACGGGGGCGATTGGCTTTATCATTGTTATGCTCTTCCTGGCGGTACGCTGGTTACAACGGCAACTTGCCGGGCAAGAATTGCTGGAAACCCGGGCTACTCGTATCTTAAACGGTGAGCGTGGCTCTAATGTGTTGGGAACCATCTATGAATGGCCGCCCAGAACCAGCAGTGCGCTGGATACGCTGCTTCGTGAAATTCAGAACGCACGCGAACAACACAGCCGTCTTGATACGCTGATCCGCTCTTATGCCGCCCAGGACGTGAAAACCGGCCTCAATAACCGACTCTTTTTCGATAATCAGTTAGCAACGTTACTGGAAGATCAGGAGAAAGTAGGTACCCACGGGATCGTGATGATGATTCGTCTGCCGGATTTCAATATGTTGAGCGATACCTGGGGGCACAGCCAGGTTGAAGAACAGTTCTTCACTCTGACGAATCTGCTGTCGACATTTATGATGCGCTACCCTGGCGCACTGCTGGCGCGTTACCACCGCAGTGATTTTGCTGCGCTGTTACCGCACCGGACGTTAAAAGAGGCAGAGAGCATCGCCGGTCAGTTAATCAAAGCCGTTGATACCTTGCCGAACAATAAAATGCTCGATCGCGACGATATGATCCACATTGGTATCTGCGCCTGGCGTAGTGGTCAGGATACCGAGCAGGTAATGGAACATGCAGAGTCTGCCACGCGTAATGCGGGATTGCAGGGCGGCAATAGCTGGGCTATTTACGATGACTCGTTGCCTGAAAAAGGACGCGGTAATGTTCGCTGGCGTACGCTTATCGAGCAAATGCTCAGTCGCGGCGGCCCGCGCCTTTATCAAAAACCGGCGGTTACTCGCGAAGGTCAGGTTCATCATCGCGAACTCATGTGCCGCATCTTCGATGGTAATGAAGAGGTTAGCTCGGCGGAGTATATGCCGATGGTCTTGCAGTTTGGCTTATCGGAAGAGTATGACCGTCTGCAAATCAGCCGTCTTATTCCACTATTGCGTTACTGGCCAGAGGAAAATCTGGCGATTCAGGTTACCGTTGAGTCGCTGATTCGCCCGCGTTTTCAGCGTTGGCTGCGCGATACGTTAATGCAATGTGAAAAATCACAACGAAAACGCATAATTATTGAACTTGCAGAGGCCGATGTAGGTCAACATATCAGTCGTTTACAACCTGTTATTCGTTTAGTGAATGCTTTAGGGGTACGGGTAGCCGTCAACCAGGCTGGTTTGACGCTGGTAAGTACCAGTTGGATCAAAGAACTTAATGTTGAGTTACTCAAGCTCCATCCGGGGCTGGTCAGAAACATTGAGAAGCGAACGGAGAACCAGCTGCTGGTTCAAAGCCTGGTGGAAGCCTGCTCCGGGACCAGCACCCAGGTTTACGCCACCGGCGTGCGTTCGCGAAGCGAGTGGCAGACCCTGATTCAGCGCGGTGTTACAGGCGGGCAAGGGGATTTTTTCGCGTCCTCACAGCCACTTGATACTAACGTGAAAAAATATTCACAAAGATACTCGGTTTAA
SEQ ID NO:6 CsrD多肽序列
MRLTTKFSAFVTLLTGLTIFVTLLGCSLSFYNAIQYKFSHRVQAVATAIDTHLVSNDFSVLRPQITELMMSADIVRVDLLHGDKQVYTLARNGSYRPVGSSDLFRELSVPLIKHPGMSLRLVYQDPMGNYFHSLMTTAPLTGAIGFIIVMLFLAVRWLQRQLAGQELLETRATRILNGERGSNVLGTIYEWPPRTSSALDTLLREIQNAREQHSRLDTLIRSYAAQDVKTGLNNRLFFDNQLATLLEDQEKVGTHGIVMMIRLPDFNMLSDTWGHSQVEEQFFTLTNLLSTFMMRYPGALLARYHRSDFAALLPHRTLKEAESIAGQLIKAVDTLPNNKMLDRDDMIHIGICAWRSGQDTEQVMEHAESATRNAGLQGGNSWAIYDDSLPEKGRGNVRWRTLIEQMLSRGGPRLYQKPAVTREGQVHHRELMCRIFDGNEEVSSAEYMPMVLQFGLSEEYDRLQISRLIPLLRYWPEENLAIQVTVESLIRPRFQRWLRDTLMQCEKSQRKRIIIELAEADVGQHISRLQPVIRLVNALGVRVAVNQAGLTLVSTSWIKELNVELLKLHPGLVRNIEKRTENQLLVQSLVEACSGTSTQVYATGVRSRSEWQTLIQRGVTGGQGDFFASSQPLDTNVKKYSQRYSV
SEQ ID NO:7 LysC多肽序列
MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVALAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALALRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVALTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFE
SEQ ID NO:8 DapA多肽序列
MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTSAIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVIAGTGANATAEAISLTQRFNDSGIVGCLTVTPYYNRPSQEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVGRLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGDDASALDFMQLGGHGVISVTANVAARDMAQMCKLAAEGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKELGLVATDTLRLPMTPITDSGRETVRAALKHAGLL
SEQ ID NO:9 LysA多肽序列
MPHSLFSTDTDLTAENLLRLPAEFGCPVWVYDAQIIRRQIAALKQFDVVRFAQKACSNIHILRLMREQGVKVDSVSLGEIERALAAGYNPQTHPDDIVFTADVIDQATLERVSELQIPVNAGSVDMLDQLGQVSPGHRVWLRVNPGFGHGHSQKTNTGGENSKHGIWYTDLPAALDVIQRHHLQLVGIHMHIGSGVDYAHLEQVCGAMVRQVIEFGQDLQAISAGGGLSVPYQQGEEAVDTEHYYGLWNAAREQIARHLGHPVKLEIEPGRFLVAQSGVLITQVRSVKQMGSRHFVLVDAGFNDLMRPAMYGSYHHISALAADGRSLEHAPTVETVVAGPLCESGDVFTQQEGGNVETRALPEVKAGDYLVLHDTGAYGASMSSNYNSRPLLPEVLFDNGQARLIRRRQTIEELLALELL
SEQ ID NO:10 LysC-1M318I,G323D多肽序列
MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVALAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALALRRNQTLLTLHSLNILHSRDFLAEVFGILARHNISVDLITTSEVSVALTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFE
SEQ ID NO:11 S-LysC多肽序列
MGLVVQKYGGSSVADAEGIKRVAKRIVEAKKNGNQVVAVVSAMGDTTDELIDLAEQVSPIPAGRELDMLLTAGERISMALLAMAIKNLGHEAQSFTGSQAGVITDSVHNKARIIDVTPGRIRTSVDEGNVAIVAGFQGVSQDSKDITTLGRGGSDTTAVALAAALDADVCEIYTDVDGVFTADPRVVPKAKKIDWISFEDMLELAASGSKVLLHRCVEYARRYNIPIHVRSSFSGLQGTWVSSEPIKQGEKHVEQALISGVAHDTSEAKVTVVGVPDKPGEAAAIFRAIADAQVNIDMVVQNVSAASTGLTDISFTLPKSEGRKAIDALEKNRPGIGFDSLRYDDQIGKISLVGAGMKSNPGVTADFFTALSDAGVNIELISTSEIRISVVTRKDDVNEAVRAVHTAFGLDSDSDEAVVYGGTGR
SEQ ID NO:12 Asd多肽序列
MKNVGFIGWRGMVGSVLMQRMVEERDFDAIRPVFFSTSQLGQAAPSFGGTTGTLQDAFDLEALKALDIIVTCQGGDYTNEIYPKLRESGWQGYWIDAASSLRMKDDAIIILDPVNQDVITDGLNNGIRTFVGGNCTVSLMLMSLGGLFANDLVDWVSVATYQAASGGGARHMRELLTQMGHLYGHVADELATPSSAILDIERKVTTLTRSGELPVDNFGVPLAGSLIPWIDKQLDNGQSREEWKGQAETNKILNTSSVIPVDGLCVRVGALRCHSQAFTIKLKKDVSIPTVEELLAAHNPWAKVVPNDREITMRELTPAAVTGTLTTPVGRLRKLNMGPEFLSAFTVGDQ
SEQ ID NO:13 DapB多肽序列
MHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIEIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNL
SEQ ID NO:14 DapD多肽序列
MQQLQNIIETAFERRAEITPANADTVTREAVNQVIALLDSGALRVAEKIDGQWVTHQWLKKAVLLSFRINDNQVIEGAESRYFDKVPMKFADYDEARFQKEGFRVVPPAAVRQGAFIARNTVLMPSYVNIGAYVDEGTMVDTWATVGSCAQIGKNVHLSGGVGIGGVLEPLQANPTIIEDNCFIGARSEVVEGVIVEEGSVISMGVYIGQSTRIYDRETGEIHYGRVPAGSVVVSGNLPSKDGKYSLYCAVIVKKVDAKTRGKVGINELLRTID
SEQ ID NO:15 AspC多肽序列
MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVLSEQ ID NO:16大肠杆菌csrB核酸序列
GAGTCAGACAACGAAGTGAACATCAGGATGATGACACTTCTGCAGGACACACCAGGATGGTGTTTCAGGGAAAGGCTTCTGGATGAAGCGAAGAGGATGACGCAGGACGCGTTAAAGGACACCTCCAGGATGGAGAATGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCAGGGACACTTCAGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAGGATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGGGAGCAACAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCGCTGTGAATACAGTGCTCCCTTTTTTTATT
SEQ ID NO:17大肠杆菌csrC核酸序列
ATAGAGCGAGGACGCTAACAGGAACAATGACTCAGGATGAGGGTCAGGAGCGCCAGGAGGCGAAGACAGAGGATTGTCAGGAAGACAAACGTCCGGAGACGTAATTAAACGGAAATGGAATCAACACGGATTGTTCCGGCTAAAGGAAAAACAGGGTGTGTTGGCGGCCTGCAAGGATTGTAAGACCCGTTAAGGGTTATGAGTCAGGAAAAAAGGCGACAGAGTAATCTGTCGCCTTTTTTCTT
SEQ ID NO:18福氏志贺菌CsrB核酸序列(CP024470.1)
GAGTCAGACAACGAAGTGAACATCAGGATGATGACACTTCTGCAGGACACACCAGGATGGTGTTTCAGGGAAAGGCTTCTGGATGAAGCGAAGAGGATGACGCAGGACGCGTTAAAGGACACCTCCAGGATGGAGAATGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCAGGGACACTTCAGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAGGATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGGGAGCAACAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCACTGTGAATACAGTGCTCCCTTTTTTTATT
SEQ ID NO:19普通小麦(Triticum aestivum)CsrB核酸序列(AK447219.1)GAGTCAGACAACGAAGTGAACATCAGGATGATGACACTTCTGCAGGAAACACCAGGATGGTGTTTCAGGGAAAGGCTTCTGGATGAAGCGAAGAGGATGACGCAGGACGCGTTAAAGGACACCTCCAGGATGGAGAATGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCAGGGACACTTCAGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAGGATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGGGAGCAACAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCGCTGTGAATACAGTGCTCCCTTTTTTTATT
SEQ ID NO:20宋氏志贺菌CsrB核酸序列(CP023645.1)
GAGTCAGACAACGAAGTGAACATCAGGATGATGACACTTCTGCAGGACACACCAGGATGGTGTTTCAGGGAAAGGCTTCTGGATGAAGCGAAGAGGATGACGCAGGACGCGTTAAAGGACACCTCCAGGATGGAGAATGAGAACCGGTCAGGATGATTCGGTGGGTCAGGAAGGCCAGGGACACTTCAGGATGAAGTATCACATCGGGGTGGTGTGAGCAGGAAGCAATAGTTCAGGATGAACGATTGGCCGCAAGGCCAGAGGAAAAGTTGTCAAGGATGAGCAGGGAGCAACAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCACTGTGAATACAGTGCTCCCTTTTTTTATT
SEQ ID NO:21柠檬酸杆菌属CsrB核酸序列(LT556085.1)
GAGTCGCACAACGAAGTGAACATCAGGATGATGACACTTCAGCAGGACACACCAGGACGGTGTTACAGGGAAAGGCTTCAGGATGAAGTGAAGAGGATAACGCGGGAATGCGTTAAAGGACACCTCCAGGAAGGAGAACGAGAGCCGGTCAGGATAGTCGGTGGGTCAGGATGGCCAGGATGCTTCAGGATGAAGTAATCACATCGGGGTGGTGTGAGCAGGATGCAAAATGTTCAGGATGAACAACAGGTCGCAAGACCAGAGGAAAAGTTGTCACGGATGAGCAGGGAGCACAAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCACTGTTTTTACAGTGCTCCCTTTTTTT
SEQ ID NO:22肠道沙门氏菌亚种CsrB核酸序列(CP015574.1)
GAGTCGTACAACGAAGCGAACGTCAGGATGATGACGCTTCAGCAGGACACGCCAGGATGGTGTTACAAGGAAAGGCTTCAGGATGAAGCAAAGTGGAAAGCGCAGGATGCGTTAAAGGACACCTCCAGGACGGAGAACGAGAGCCGATCAGGATGTTCGGCGGGTCTGGATGACCAGGGACGCTTCAGGATGAAGCTATCACATCGGGCGATGTGCGCAGGATGCAAACGTTCAGGATGAACAGGCCGTAAGGTCACAGGAAAAGTTGTCACGGATGAGCAGGGAGCACGAAAAGTAGCTGGAATGCTGCGAAACGAACCGGGAGCACTGTTTATACAGTGCTCCCTTTTTTT
SEQ ID NO:23宋氏志贺菌CsrC核酸序列(CP023645.1)
ATAGAGCGAGGACGCTAACAGGAACAATGACTCAGGATGAGGGTCAGGAGCGCCAGGAGGCGAAGACAGAGGATTGTCAGGAAGACAAACGTCCGGAGACGTAATTAAACGGAAATGGAATCAACACGGATTGTTCCGGCTAAAGGAAAAACAGGGTGTGTTGGCGGCCTGCAAGGATTGTAAGACCCGTTAAGGGTTATGAGTCAGGAAAAAAGGCGACAGAGTAATCTGTCGCCTTTTTTCTT
SEQ ID NO:24福氏志贺菌CsrC核酸序列(CP024470.1)
ATAGAGCGAGGACGCTAACAGGAACAATGACTCAGGATGAGGGTCAGGAGCGCCAGGAGGCGAAGACAGAGGATTGTCAGGAAGACAAACGTCCGGAGACGTAATTAAACGGAAATGGAATCAACACGGATTGTTCCGGCTAATGGAAAAACAGGGTGTGTTGGCGGCCTGCAAGGATTGTAAGACCCGTTAAGGGTTATGAGTCAGGAAAAAAGGCGACAGAGTAATCTGTCGCCTTTTTTCTT
SEQ ID NO:25韦氏柠檬酸杆菌CsrC核酸序列(CP023504.1)
AGCGAGGGAGCTAACAGGATAAACGACTCAGGATGAGGGTCAGGAGCGCCAGGAGGCGAAGACGCAGGATTGTCAGGAAGACAAACGTCCGGAGACGTTAGTAAAAGGAAATGGAAACAACACGGAATGTTTCTGGCTAAGGGAAAAACAGGGTGTGTTGATAGCCGACAGGGATTGTAGGGCCCGTTAAGGGTTGAGAGTCAGGAAAAAAGGCGACAGATTACTCTGTCGCCTTTTTTCTT
SEQ ID NO:26肠道沙门氏菌亚种CsrC核酸序列(CP018661.1)
AGCGAGGACGCTAACAGGATCAACGACTCAGGATGAGGGTCAGGAGCGCCAGGAGGCGAAGACACAGGATTGTCAGGAAGACAAACGTCCGGAGACGTTAGTAAAAGGAAATGGAAACAACATGGAATGTTCCAGGCTAAGGGAAAAACAGGGCGTGTTGATAGCCAACAGGGATGGTGGAACCCGTTAAGGGTCGTGAGTCAAGAAAAAAGGCGGCAGATTACTCTGTCGCCTTTTTTCTT
序列表
<110> 上海凯赛生物技术股份有限公司
CIBT美国公司
<120> 调节CSR系统以生产赖氨酸和赖氨酸衍生产物
<130> IBPA17007
<160> 76
<170> PatentIn version 3.5
<210> 1
<211> 2148
<212> DNA
<213> Escherichia coli
<400> 1
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 2
<211> 715
<212> PRT
<213> Escherichia coli
<400> 2
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 3
<211> 186
<212> DNA
<213> Escherichia coli
<400> 3
atgctgattc tgactcgtcg agttggtgag accctcatga ttggggatga ggtcaccgtg 60
acagttttag gggtaaaggg caaccaggta cgtattggcg taaatgcccc gaaggaagtt 120
tctgttcacc gtgaagagat ctaccagcgt atccaggctg aaaaatccca gcagtccagt 180
tactaa 186
<210> 4
<211> 61
<212> PRT
<213> Escherichia coli
<400> 4
Met Leu Ile Leu Thr Arg Arg Val Gly Glu Thr Leu Met Ile Gly Asp
1 5 10 15
Glu Val Thr Val Thr Val Leu Gly Val Lys Gly Asn Gln Val Arg Ile
20 25 30
Gly Val Asn Ala Pro Lys Glu Val Ser Val His Arg Glu Glu Ile Tyr
35 40 45
Gln Arg Ile Gln Ala Glu Lys Ser Gln Gln Ser Ser Tyr
50 55 60
<210> 5
<211> 1941
<212> DNA
<213> Escherichia coli
<400> 5
atgagattaa cgacgaaatt ttcggccttt gttacgctgc tcaccgggtt aacaattttt 60
gtgactttgc tgggctgttc gctaagtttc tacaacgcca ttcagtataa gtttagtcat 120
cgcgttcagg cggtggcgac ggcgattgat acccaccttg tgtcgaatga cttcagcgta 180
ttaaggccac aaattaccga attaatgatg tcggcagata tcgttcgtgt agacctgctc 240
catggtgata aacaggttta taccctggcc agaaatggta gttatcgtcc agttggctcc 300
agcgatctgt ttcgcgaact gagcgttccg ttgataaagc atccggggat gtcgttgcgt 360
ctggtttatc aggacccgat gggcaactat ttccattcgt tgatgaccac cgcgccgctc 420
acgggggcga ttggctttat cattgttatg ctcttcctgg cggtacgctg gttacaacgg 480
caacttgccg ggcaagaatt gctggaaacc cgggctactc gtatcttaaa cggtgagcgt 540
ggctctaatg tgttgggaac catctatgaa tggccgccca gaaccagcag tgcgctggat 600
acgctgcttc gtgaaattca gaacgcacgc gaacaacaca gccgtcttga tacgctgatc 660
cgctcttatg ccgcccagga cgtgaaaacc ggcctcaata accgactctt tttcgataat 720
cagttagcaa cgttactgga agatcaggag aaagtaggta cccacgggat cgtgatgatg 780
attcgtctgc cggatttcaa tatgttgagc gatacctggg ggcacagcca ggttgaagaa 840
cagttcttca ctctgacgaa tctgctgtcg acatttatga tgcgctaccc tggcgcactg 900
ctggcgcgtt accaccgcag tgattttgct gcgctgttac cgcaccggac gttaaaagag 960
gcagagagca tcgccggtca gttaatcaaa gccgttgata ccttgccgaa caataaaatg 1020
ctcgatcgcg acgatatgat ccacattggt atctgcgcct ggcgtagtgg tcaggatacc 1080
gagcaggtaa tggaacatgc agagtctgcc acgcgtaatg cgggattgca gggcggcaat 1140
agctgggcta tttacgatga ctcgttgcct gaaaaaggac gcggtaatgt tcgctggcgt 1200
acgcttatcg agcaaatgct cagtcgcggc ggcccgcgcc tttatcaaaa accggcggtt 1260
actcgcgaag gtcaggttca tcatcgcgaa ctcatgtgcc gcatcttcga tggtaatgaa 1320
gaggttagct cggcggagta tatgccgatg gtcttgcagt ttggcttatc ggaagagtat 1380
gaccgtctgc aaatcagccg tcttattcca ctattgcgtt actggccaga ggaaaatctg 1440
gcgattcagg ttaccgttga gtcgctgatt cgcccgcgtt ttcagcgttg gctgcgcgat 1500
acgttaatgc aatgtgaaaa atcacaacga aaacgcataa ttattgaact tgcagaggcc 1560
gatgtaggtc aacatatcag tcgtttacaa cctgttattc gtttagtgaa tgctttaggg 1620
gtacgggtag ccgtcaacca ggctggtttg acgctggtaa gtaccagttg gatcaaagaa 1680
cttaatgttg agttactcaa gctccatccg gggctggtca gaaacattga gaagcgaacg 1740
gagaaccagc tgctggttca aagcctggtg gaagcctgct ccgggaccag cacccaggtt 1800
tacgccaccg gcgtgcgttc gcgaagcgag tggcagaccc tgattcagcg cggtgttaca 1860
ggcgggcaag gggatttttt cgcgtcctca cagccacttg atactaacgt gaaaaaatat 1920
tcacaaagat actcggttta a 1941
<210> 6
<211> 646
<212> PRT
<213> Escherichia coli
<400> 6
Met Arg Leu Thr Thr Lys Phe Ser Ala Phe Val Thr Leu Leu Thr Gly
1 5 10 15
Leu Thr Ile Phe Val Thr Leu Leu Gly Cys Ser Leu Ser Phe Tyr Asn
20 25 30
Ala Ile Gln Tyr Lys Phe Ser His Arg Val Gln Ala Val Ala Thr Ala
35 40 45
Ile Asp Thr His Leu Val Ser Asn Asp Phe Ser Val Leu Arg Pro Gln
50 55 60
Ile Thr Glu Leu Met Met Ser Ala Asp Ile Val Arg Val Asp Leu Leu
65 70 75 80
His Gly Asp Lys Gln Val Tyr Thr Leu Ala Arg Asn Gly Ser Tyr Arg
85 90 95
Pro Val Gly Ser Ser Asp Leu Phe Arg Glu Leu Ser Val Pro Leu Ile
100 105 110
Lys His Pro Gly Met Ser Leu Arg Leu Val Tyr Gln Asp Pro Met Gly
115 120 125
Asn Tyr Phe His Ser Leu Met Thr Thr Ala Pro Leu Thr Gly Ala Ile
130 135 140
Gly Phe Ile Ile Val Met Leu Phe Leu Ala Val Arg Trp Leu Gln Arg
145 150 155 160
Gln Leu Ala Gly Gln Glu Leu Leu Glu Thr Arg Ala Thr Arg Ile Leu
165 170 175
Asn Gly Glu Arg Gly Ser Asn Val Leu Gly Thr Ile Tyr Glu Trp Pro
180 185 190
Pro Arg Thr Ser Ser Ala Leu Asp Thr Leu Leu Arg Glu Ile Gln Asn
195 200 205
Ala Arg Glu Gln His Ser Arg Leu Asp Thr Leu Ile Arg Ser Tyr Ala
210 215 220
Ala Gln Asp Val Lys Thr Gly Leu Asn Asn Arg Leu Phe Phe Asp Asn
225 230 235 240
Gln Leu Ala Thr Leu Leu Glu Asp Gln Glu Lys Val Gly Thr His Gly
245 250 255
Ile Val Met Met Ile Arg Leu Pro Asp Phe Asn Met Leu Ser Asp Thr
260 265 270
Trp Gly His Ser Gln Val Glu Glu Gln Phe Phe Thr Leu Thr Asn Leu
275 280 285
Leu Ser Thr Phe Met Met Arg Tyr Pro Gly Ala Leu Leu Ala Arg Tyr
290 295 300
His Arg Ser Asp Phe Ala Ala Leu Leu Pro His Arg Thr Leu Lys Glu
305 310 315 320
Ala Glu Ser Ile Ala Gly Gln Leu Ile Lys Ala Val Asp Thr Leu Pro
325 330 335
Asn Asn Lys Met Leu Asp Arg Asp Asp Met Ile His Ile Gly Ile Cys
340 345 350
Ala Trp Arg Ser Gly Gln Asp Thr Glu Gln Val Met Glu His Ala Glu
355 360 365
Ser Ala Thr Arg Asn Ala Gly Leu Gln Gly Gly Asn Ser Trp Ala Ile
370 375 380
Tyr Asp Asp Ser Leu Pro Glu Lys Gly Arg Gly Asn Val Arg Trp Arg
385 390 395 400
Thr Leu Ile Glu Gln Met Leu Ser Arg Gly Gly Pro Arg Leu Tyr Gln
405 410 415
Lys Pro Ala Val Thr Arg Glu Gly Gln Val His His Arg Glu Leu Met
420 425 430
Cys Arg Ile Phe Asp Gly Asn Glu Glu Val Ser Ser Ala Glu Tyr Met
435 440 445
Pro Met Val Leu Gln Phe Gly Leu Ser Glu Glu Tyr Asp Arg Leu Gln
450 455 460
Ile Ser Arg Leu Ile Pro Leu Leu Arg Tyr Trp Pro Glu Glu Asn Leu
465 470 475 480
Ala Ile Gln Val Thr Val Glu Ser Leu Ile Arg Pro Arg Phe Gln Arg
485 490 495
Trp Leu Arg Asp Thr Leu Met Gln Cys Glu Lys Ser Gln Arg Lys Arg
500 505 510
Ile Ile Ile Glu Leu Ala Glu Ala Asp Val Gly Gln His Ile Ser Arg
515 520 525
Leu Gln Pro Val Ile Arg Leu Val Asn Ala Leu Gly Val Arg Val Ala
530 535 540
Val Asn Gln Ala Gly Leu Thr Leu Val Ser Thr Ser Trp Ile Lys Glu
545 550 555 560
Leu Asn Val Glu Leu Leu Lys Leu His Pro Gly Leu Val Arg Asn Ile
565 570 575
Glu Lys Arg Thr Glu Asn Gln Leu Leu Val Gln Ser Leu Val Glu Ala
580 585 590
Cys Ser Gly Thr Ser Thr Gln Val Tyr Ala Thr Gly Val Arg Ser Arg
595 600 605
Ser Glu Trp Gln Thr Leu Ile Gln Arg Gly Val Thr Gly Gly Gln Gly
610 615 620
Asp Phe Phe Ala Ser Ser Gln Pro Leu Asp Thr Asn Val Lys Lys Tyr
625 630 635 640
Ser Gln Arg Tyr Ser Val
645
<210> 7
<211> 449
<212> PRT
<213> Escherichia coli
<400> 7
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 8
<211> 292
<212> PRT
<213> Escherichia coli
<400> 8
Met Phe Thr Gly Ser Ile Val Ala Ile Val Thr Pro Met Asp Glu Lys
1 5 10 15
Gly Asn Val Cys Arg Ala Ser Leu Lys Lys Leu Ile Asp Tyr His Val
20 25 30
Ala Ser Gly Thr Ser Ala Ile Val Ser Val Gly Thr Thr Gly Glu Ser
35 40 45
Ala Thr Leu Asn His Asp Glu His Ala Asp Val Val Met Met Thr Leu
50 55 60
Asp Leu Ala Asp Gly Arg Ile Pro Val Ile Ala Gly Thr Gly Ala Asn
65 70 75 80
Ala Thr Ala Glu Ala Ile Ser Leu Thr Gln Arg Phe Asn Asp Ser Gly
85 90 95
Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro Ser Gln
100 105 110
Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu His Thr Asp Leu
115 120 125
Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg Thr Gly Cys Asp Leu Leu
130 135 140
Pro Glu Thr Val Gly Arg Leu Ala Lys Val Lys Asn Ile Ile Gly Ile
145 150 155 160
Lys Glu Ala Thr Gly Asn Leu Thr Arg Val Asn Gln Ile Lys Glu Leu
165 170 175
Val Ser Asp Asp Phe Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu
180 185 190
Asp Phe Met Gln Leu Gly Gly His Gly Val Ile Ser Val Thr Ala Asn
195 200 205
Val Ala Ala Arg Asp Met Ala Gln Met Cys Lys Leu Ala Ala Glu Gly
210 215 220
His Phe Ala Glu Ala Arg Val Ile Asn Gln Arg Leu Met Pro Leu His
225 230 235 240
Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pro Val Lys Trp Ala Cys
245 250 255
Lys Glu Leu Gly Leu Val Ala Thr Asp Thr Leu Arg Leu Pro Met Thr
260 265 270
Pro Ile Thr Asp Ser Gly Arg Glu Thr Val Arg Ala Ala Leu Lys His
275 280 285
Ala Gly Leu Leu
290
<210> 9
<211> 420
<212> PRT
<213> Escherichia coli
<400> 9
Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn
1 5 10 15
Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp
20 25 30
Ala Gln Ile Ile Arg Arg Gln Ile Ala Ala Leu Lys Gln Phe Asp Val
35 40 45
Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu
50 55 60
Met Arg Glu Gln Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile
65 70 75 80
Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp
85 90 95
Ile Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg Val
100 105 110
Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp
115 120 125
Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn
130 135 140
Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu
145 150 155 160
Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp
165 170 175
Val Ile Gln Arg His His Leu Gln Leu Val Gly Ile His Met His Ile
180 185 190
Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met
195 200 205
Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala
210 215 220
Gly Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp
225 230 235 240
Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile Ala
245 250 255
Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe
260 265 270
Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln Val Arg Ser Val Lys
275 280 285
Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp
290 295 300
Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu
305 310 315 320
Ala Ala Asp Gly Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val
325 330 335
Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu
340 345 350
Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp
355 360 365
Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser
370 375 380
Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly
385 390 395 400
Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu Leu Leu Ala
405 410 415
Leu Glu Leu Leu
420
<210> 10
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> LysC-1 M318I, G323D polypeptide sequence
<400> 10
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Ile Leu His
305 310 315 320
Ser Arg Asp Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 11
<211> 425
<212> PRT
<213> Artificial Sequence
<220>
<223> S-LysC polypeptide sequence
<400> 11
Met Gly Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Val Ala Asp Ala
1 5 10 15
Glu Gly Ile Lys Arg Val Ala Lys Arg Ile Val Glu Ala Lys Lys Asn
20 25 30
Gly Asn Gln Val Val Ala Val Val Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Ile Asp Leu Ala Glu Gln Val Ser Pro Ile Pro Ala Gly Arg
50 55 60
Glu Leu Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Met Ala Leu
65 70 75 80
Leu Ala Met Ala Ile Lys Asn Leu Gly His Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Ile Thr Asp Ser Val His Asn Lys Ala Arg
100 105 110
Ile Ile Asp Val Thr Pro Gly Arg Ile Arg Thr Ser Val Asp Glu Gly
115 120 125
Asn Val Ala Ile Val Ala Gly Phe Gln Gly Val Ser Gln Asp Ser Lys
130 135 140
Asp Ile Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asp Ala Asp Val Cys Glu Ile Tyr Thr Asp Val
165 170 175
Asp Gly Val Phe Thr Ala Asp Pro Arg Val Val Pro Lys Ala Lys Lys
180 185 190
Ile Asp Trp Ile Ser Phe Glu Asp Met Leu Glu Leu Ala Ala Ser Gly
195 200 205
Ser Lys Val Leu Leu His Arg Cys Val Glu Tyr Ala Arg Arg Tyr Asn
210 215 220
Ile Pro Ile His Val Arg Ser Ser Phe Ser Gly Leu Gln Gly Thr Trp
225 230 235 240
Val Ser Ser Glu Pro Ile Lys Gln Gly Glu Lys His Val Glu Gln Ala
245 250 255
Leu Ile Ser Gly Val Ala His Asp Thr Ser Glu Ala Lys Val Thr Val
260 265 270
Val Gly Val Pro Asp Lys Pro Gly Glu Ala Ala Ala Ile Phe Arg Ala
275 280 285
Ile Ala Asp Ala Gln Val Asn Ile Asp Met Val Val Gln Asn Val Ser
290 295 300
Ala Ala Ser Thr Gly Leu Thr Asp Ile Ser Phe Thr Leu Pro Lys Ser
305 310 315 320
Glu Gly Arg Lys Ala Ile Asp Ala Leu Glu Lys Asn Arg Pro Gly Ile
325 330 335
Gly Phe Asp Ser Leu Arg Tyr Asp Asp Gln Ile Gly Lys Ile Ser Leu
340 345 350
Val Gly Ala Gly Met Lys Ser Asn Pro Gly Val Thr Ala Asp Phe Phe
355 360 365
Thr Ala Leu Ser Asp Ala Gly Val Asn Ile Glu Leu Ile Ser Thr Ser
370 375 380
Glu Ile Arg Ile Ser Val Val Thr Arg Lys Asp Asp Val Asn Glu Ala
385 390 395 400
Val Arg Ala Val His Thr Ala Phe Gly Leu Asp Ser Asp Ser Asp Glu
405 410 415
Ala Val Val Tyr Gly Gly Thr Gly Arg
420 425
<210> 12
<211> 350
<212> PRT
<213> Escherichia coli
<400> 12
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Glu Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln
340 345 350
<210> 13
<211> 273
<212> PRT
<213> Escherichia coli
<400> 13
Met His Asp Ala Asn Ile Arg Val Ala Ile Ala Gly Ala Gly Gly Arg
1 5 10 15
Met Gly Arg Gln Leu Ile Gln Ala Ala Leu Ala Leu Glu Gly Val Gln
20 25 30
Leu Gly Ala Ala Leu Glu Arg Glu Gly Ser Ser Leu Leu Gly Ser Asp
35 40 45
Ala Gly Glu Leu Ala Gly Ala Gly Lys Thr Gly Val Thr Val Gln Ser
50 55 60
Ser Leu Asp Ala Val Lys Asp Asp Phe Asp Val Phe Ile Asp Phe Thr
65 70 75 80
Arg Pro Glu Gly Thr Leu Asn His Leu Ala Phe Cys Arg Gln His Gly
85 90 95
Lys Gly Met Val Ile Gly Thr Thr Gly Phe Asp Glu Ala Gly Lys Gln
100 105 110
Ala Ile Arg Asp Ala Ala Ala Asp Ile Ala Ile Val Phe Ala Ala Asn
115 120 125
Phe Ser Val Gly Val Asn Val Met Leu Lys Leu Leu Glu Lys Ala Ala
130 135 140
Lys Val Met Gly Asp Tyr Thr Asp Ile Glu Ile Ile Glu Ala His His
145 150 155 160
Arg His Lys Val Asp Ala Pro Ser Gly Thr Ala Leu Ala Met Gly Glu
165 170 175
Ala Ile Ala His Ala Leu Asp Lys Asp Leu Lys Asp Cys Ala Val Tyr
180 185 190
Ser Arg Glu Gly His Thr Gly Glu Arg Val Pro Gly Thr Ile Gly Phe
195 200 205
Ala Thr Val Arg Ala Gly Asp Ile Val Gly Glu His Thr Ala Met Phe
210 215 220
Ala Asp Ile Gly Glu Arg Leu Glu Ile Thr His Lys Ala Ser Ser Arg
225 230 235 240
Met Thr Phe Ala Asn Gly Ala Val Arg Ser Ala Leu Trp Leu Ser Gly
245 250 255
Lys Glu Ser Gly Leu Phe Asp Met Arg Asp Val Leu Asp Leu Asn Asn
260 265 270
Leu
<210> 14
<211> 274
<212> PRT
<213> Escherichia coli
<400> 14
Met Gln Gln Leu Gln Asn Ile Ile Glu Thr Ala Phe Glu Arg Arg Ala
1 5 10 15
Glu Ile Thr Pro Ala Asn Ala Asp Thr Val Thr Arg Glu Ala Val Asn
20 25 30
Gln Val Ile Ala Leu Leu Asp Ser Gly Ala Leu Arg Val Ala Glu Lys
35 40 45
Ile Asp Gly Gln Trp Val Thr His Gln Trp Leu Lys Lys Ala Val Leu
50 55 60
Leu Ser Phe Arg Ile Asn Asp Asn Gln Val Ile Glu Gly Ala Glu Ser
65 70 75 80
Arg Tyr Phe Asp Lys Val Pro Met Lys Phe Ala Asp Tyr Asp Glu Ala
85 90 95
Arg Phe Gln Lys Glu Gly Phe Arg Val Val Pro Pro Ala Ala Val Arg
100 105 110
Gln Gly Ala Phe Ile Ala Arg Asn Thr Val Leu Met Pro Ser Tyr Val
115 120 125
Asn Ile Gly Ala Tyr Val Asp Glu Gly Thr Met Val Asp Thr Trp Ala
130 135 140
Thr Val Gly Ser Cys Ala Gln Ile Gly Lys Asn Val His Leu Ser Gly
145 150 155 160
Gly Val Gly Ile Gly Gly Val Leu Glu Pro Leu Gln Ala Asn Pro Thr
165 170 175
Ile Ile Glu Asp Asn Cys Phe Ile Gly Ala Arg Ser Glu Val Val Glu
180 185 190
Gly Val Ile Val Glu Glu Gly Ser Val Ile Ser Met Gly Val Tyr Ile
195 200 205
Gly Gln Ser Thr Arg Ile Tyr Asp Arg Glu Thr Gly Glu Ile His Tyr
210 215 220
Gly Arg Val Pro Ala Gly Ser Val Val Val Ser Gly Asn Leu Pro Ser
225 230 235 240
Lys Asp Gly Lys Tyr Ser Leu Tyr Cys Ala Val Ile Val Lys Lys Val
245 250 255
Asp Ala Lys Thr Arg Gly Lys Val Gly Ile Asn Glu Leu Leu Arg Thr
260 265 270
Ile Asp
<210> 15
<211> 396
<212> PRT
<213> Escherichia coli
<400> 15
Met Phe Glu Asn Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Leu
1 5 10 15
Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly
20 25 30
Ile Gly Val Tyr Lys Asp Glu Thr Gly Lys Thr Pro Val Leu Thr Ser
35 40 45
Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn
50 55 60
Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu
65 70 75 80
Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg
85 90 95
Thr Ala Gln Thr Pro Gly Gly Thr Gly Ala Leu Arg Val Ala Ala Asp
100 105 110
Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro
115 120 125
Ser Trp Pro Asn His Lys Ser Val Phe Asn Ser Ala Gly Leu Glu Val
130 135 140
Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Leu Asp Phe Asp
145 150 155 160
Ala Leu Ile Asn Ser Leu Asn Glu Ala Gln Ala Gly Asp Val Val Leu
165 170 175
Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu
180 185 190
Gln Trp Gln Thr Leu Ala Gln Leu Ser Val Glu Lys Gly Trp Leu Pro
195 200 205
Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp
210 215 220
Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val
225 230 235 240
Ala Ser Ser Tyr Ser Lys Asn Phe Gly Leu Tyr Asn Glu Arg Val Gly
245 250 255
Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe
260 265 270
Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala
275 280 285
His Gly Ala Ser Val Val Ala Thr Ile Leu Ser Asn Asp Ala Leu Arg
290 295 300
Ala Ile Trp Glu Gln Glu Leu Thr Asp Met Arg Gln Arg Ile Gln Arg
305 310 315 320
Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg
325 330 335
Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly
340 345 350
Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr
355 360 365
Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn
370 375 380
Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu
385 390 395
<210> 16
<211> 360
<212> DNA
<213> Escherichia coli
<400> 16
gagtcagaca acgaagtgaa catcaggatg atgacacttc tgcaggacac accaggatgg 60
tgtttcaggg aaaggcttct ggatgaagcg aagaggatga cgcaggacgc gttaaaggac 120
acctccagga tggagaatga gaaccggtca ggatgattcg gtgggtcagg aaggccaggg 180
acacttcagg atgaagtatc acatcggggt ggtgtgagca ggaagcaata gttcaggatg 240
aacgattggc cgcaaggcca gaggaaaagt tgtcaaggat gagcagggag caacaaaagt 300
agctggaatg ctgcgaaacg aaccgggagc gctgtgaata cagtgctccc tttttttatt 360
<210> 17
<211> 245
<212> DNA
<213> Escherichia coli
<400> 17
atagagcgag gacgctaaca ggaacaatga ctcaggatga gggtcaggag cgccaggagg 60
cgaagacaga ggattgtcag gaagacaaac gtccggagac gtaattaaac ggaaatggaa 120
tcaacacgga ttgttccggc taaaggaaaa acagggtgtg ttggcggcct gcaaggattg 180
taagacccgt taagggttat gagtcaggaa aaaaggcgac agagtaatct gtcgcctttt 240
ttctt 245
<210> 18
<211> 360
<212> DNA
<213> Shigella flexneri
<400> 18
gagtcagaca acgaagtgaa catcaggatg atgacacttc tgcaggacac accaggatgg 60
tgtttcaggg aaaggcttct ggatgaagcg aagaggatga cgcaggacgc gttaaaggac 120
acctccagga tggagaatga gaaccggtca ggatgattcg gtgggtcagg aaggccaggg 180
acacttcagg atgaagtatc acatcggggt ggtgtgagca ggaagcaata gttcaggatg 240
aacgattggc cgcaaggcca gaggaaaagt tgtcaaggat gagcagggag caacaaaagt 300
agctggaatg ctgcgaaacg aaccgggagc actgtgaata cagtgctccc tttttttatt 360
<210> 19
<211> 360
<212> DNA
<213> Triticum aestivum
<400> 19
gagtcagaca acgaagtgaa catcaggatg atgacacttc tgcaggaaac accaggatgg 60
tgtttcaggg aaaggcttct ggatgaagcg aagaggatga cgcaggacgc gttaaaggac 120
acctccagga tggagaatga gaaccggtca ggatgattcg gtgggtcagg aaggccaggg 180
acacttcagg atgaagtatc acatcggggt ggtgtgagca ggaagcaata gttcaggatg 240
aacgattggc cgcaaggcca gaggaaaagt tgtcaaggat gagcagggag caacaaaagt 300
agctggaatg ctgcgaaacg aaccgggagc gctgtgaata cagtgctccc tttttttatt 360
<210> 20
<211> 360
<212> DNA
<213> Shigella sonnei
<400> 20
gagtcagaca acgaagtgaa catcaggatg atgacacttc tgcaggacac accaggatgg 60
tgtttcaggg aaaggcttct ggatgaagcg aagaggatga cgcaggacgc gttaaaggac 120
acctccagga tggagaatga gaaccggtca ggatgattcg gtgggtcagg aaggccaggg 180
acacttcagg atgaagtatc acatcggggt ggtgtgagca ggaagcaata gttcaggatg 240
aacgattggc cgcaaggcca gaggaaaagt tgtcaaggat gagcagggag caacaaaagt 300
agctggaatg ctgcgaaacg aaccgggagc actgtgaata cagtgctccc tttttttatt 360
<210> 21
<211> 358
<212> DNA
<213> Citrobacter sp.
<400> 21
gagtcgcaca acgaagtgaa catcaggatg atgacacttc agcaggacac accaggacgg 60
tgttacaggg aaaggcttca ggatgaagtg aagaggataa cgcgggaatg cgttaaagga 120
cacctccagg aaggagaacg agagccggtc aggatagtcg gtgggtcagg atggccagga 180
tgcttcagga tgaagtaatc acatcggggt ggtgtgagca ggatgcaaaa tgttcaggat 240
gaacaacagg tcgcaagacc agaggaaaag ttgtcacgga tgagcaggga gcacaaaaag 300
tagctggaat gctgcgaaac gaaccgggag cactgttttt acagtgctcc cttttttt 358
<210> 22
<211> 353
<212> DNA
<213> Salmonella enterica
<400> 22
gagtcgtaca acgaagcgaa cgtcaggatg atgacgcttc agcaggacac gccaggatgg 60
tgttacaagg aaaggcttca ggatgaagca aagtggaaag cgcaggatgc gttaaaggac 120
acctccagga cggagaacga gagccgatca ggatgttcgg cgggtctgga tgaccaggga 180
cgcttcagga tgaagctatc acatcgggcg atgtgcgcag gatgcaaacg ttcaggatga 240
acaggccgta aggtcacagg aaaagttgtc acggatgagc agggagcacg aaaagtagct 300
ggaatgctgc gaaacgaacc gggagcactg tttatacagt gctccctttt ttt 353
<210> 23
<211> 245
<212> DNA
<213> Shigella sonnei
<400> 23
atagagcgag gacgctaaca ggaacaatga ctcaggatga gggtcaggag cgccaggagg 60
cgaagacaga ggattgtcag gaagacaaac gtccggagac gtaattaaac ggaaatggaa 120
tcaacacgga ttgttccggc taaaggaaaa acagggtgtg ttggcggcct gcaaggattg 180
taagacccgt taagggttat gagtcaggaa aaaaggcgac agagtaatct gtcgcctttt 240
ttctt 245
<210> 24
<211> 245
<212> DNA
<213> Shigella flexneri
<400> 24
atagagcgag gacgctaaca ggaacaatga ctcaggatga gggtcaggag cgccaggagg 60
cgaagacaga ggattgtcag gaagacaaac gtccggagac gtaattaaac ggaaatggaa 120
tcaacacgga ttgttccggc taatggaaaa acagggtgtg ttggcggcct gcaaggattg 180
taagacccgt taagggttat gagtcaggaa aaaaggcgac agagtaatct gtcgcctttt 240
ttctt 245
<210> 25
<211> 242
<212> DNA
<213> Citrobacter werkmaniii
<400> 25
agcgagggag ctaacaggat aaacgactca ggatgagggt caggagcgcc aggaggcgaa 60
gacgcaggat tgtcaggaag acaaacgtcc ggagacgtta gtaaaaggaa atggaaacaa 120
cacggaatgt ttctggctaa gggaaaaaca gggtgtgttg atagccgaca gggattgtag 180
ggcccgttaa gggttgagag tcaggaaaaa aggcgacaga ttactctgtc gccttttttc 240
tt 242
<210> 26
<211> 242
<212> DNA
<213> Salmonella enterica
<400> 26
agcgaggacg ctaacaggat caacgactca ggatgagggt caggagcgcc aggaggcgaa 60
gacacaggat tgtcaggaag acaaacgtcc ggagacgtta gtaaaaggaa atggaaacaa 120
catggaatgt tccaggctaa gggaaaaaca gggcgtgttg atagccaaca gggatggtgg 180
aacccgttaa gggtcgtgag tcaagaaaaa aggcggcaga ttactctgtc gccttttttc 240
tt 242
<210> 27
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer cadA-F
<400> 27
ggcgagctca cacaggaaac agaccatgaa cgttattgca atattgaatc 50
<210> 28
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer cadA-R
<400> 28
ggcggatccc cacttccctt gtacgagcta attatttttt gctttcttct ttc 53
<210> 29
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer cadA-F2
<400> 29
atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaatcac 47
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer cadA-R2
<400> 30
agctgtttcc tgtgtgaaat 20
<210> 31
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SacI-F
<400> 31
ggcgagctcc tcctgtgtga aattgttatc cgctc 35
<210> 32
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SacI-R
<400> 32
ggcgagctca tgaacgttat tgcaatattg aatc 34
<210> 33
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrA-F
<400> 33
ggcgagctca tgctgattct gactcgtcg 29
<210> 34
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrA-R
<400> 34
ggcggatcct tagtaactgg actgctggga t 31
<210> 35
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrD-F
<400> 35
ggcgagctca tgagattaac gacgaaattt tcg 33
<210> 36
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrD-R
<400> 36
ggcggatcct taaaccgagt atctttgtga atat 34
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer rmvBamHI-F
<400> 37
gtttatcagg acccgatggg ca 22
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer rmvBamHI-R
<400> 38
tgcccatcgg gtcctgataa ac 22
<210> 39
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysC-F
<400> 39
ggcgagctca cacaggaaac agaccatgtc tgaaattgtt gtctcc 46
<210> 40
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysC-R
<400> 40
ggcggatcct tactcaaaca aattactatg cag 33
<210> 41
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapA-F
<400> 41
ggcggatcca cacaggaaac agaccatgtt cacgggaagt attgtc 46
<210> 42
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapA-R
<400> 42
ggctctagat tacagcaaac cggcatgc 28
<210> 43
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysA-F
<400> 43
ggctctagaa cacaggaaac agaccatgcc acattcactg ttcagc 46
<210> 44
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysA-R
<400> 44
ggcgtcgact taaagcaatt ccagcgccag 30
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer 318-F
<400> 45
cagcctgaat atactgcatt ctc 23
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer 318-R
<400> 46
gagaatgcag tatattcagg ctg 23
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer 323-F
<400> 47
gcattctcgc gatttcctcg 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer 323-R
<400> 48
cgaggaaatc gcgagaatgc 20
<210> 49
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SlysC-F
<400> 49
ggcgagctca cacaggaaac agaccatggg cttagttgtg cagaaa 46
<210> 50
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SlysC-R
<400> 50
ggcggatcct taacgacctg tgccgccata 30
<210> 51
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer asd-F
<400> 51
ggcgagctca cacaggaaac agaccatgaa aaatgttggt tttatcgg 48
<210> 52
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer asd-R
<400> 52
ggcggatcct tacgccagtt gacgaagc 28
<210> 53
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapB-F
<400> 53
ggcacacagg aaacagacca tgcatgatgc aaacatccg 39
<210> 54
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapB-R
<400> 54
ggctctagat tacaaattat tgagatcaag tacatctc 38
<210> 55
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapD-F
<400> 55
ggctctagaa cacaggaaac agaccatgca gcagttacag aacat 45
<210> 56
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer dapD-R
<400> 56
ggcgcatgct tagtcgatgg tacgcagca 29
<210> 57
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer aspC-F
<400> 57
ggctctagaa cacaggaaac agaccatgtt tgagaacatt accgcc 46
<210> 58
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer aspC-R
<400> 58
ggcgcatgcg acctcgaggt agtcgactta cagcactgcc acaatcg 47
<210> 59
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysC-rbs2-F
<400> 59
atttcacaca ggaaacagct atgtctgaaa ttgttgtctc ca 42
<210> 60
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer lysC-rbs2-R
<400> 60
agctgtttcc tgtgtgaaat 20
<210> 61
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer asd-rbs2-F
<400> 61
atttcacaca ggaaacagct atgaaaaatg ttggttttat cggctg 46
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer asd-rbs2-R
<400> 62
agctgtttcc tgtgtgaaat 20
<210> 63
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SacI-F2
<400> 63
ggcgagctct cccctgattc tgtggataa 29
<210> 64
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer SacI-R2
<400> 64
ggcgagctca gcaaaaggcc aggaaccgt 29
<210> 65
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer ApaI-F
<400> 65
ggcgggcccg tattaccgcc tttgagtgag 30
<210> 66
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer ApaI-R
<400> 66
ggcgggccca cagaatcagg ggagagctc 29
<210> 67
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer LAL2-SacI-F
<400> 67
ggcgagctcg ttggccgatt cattaatgc 29
<210> 68
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer LAL2-ApaI-R
<400> 68
ggcgggccct taaagcaatt ccagcgccag 30
<210> 69
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrB-F
<400> 69
ggcgagctcg agtcagacaa cgaagtgaac 30
<210> 70
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrB-R
<400> 70
ggcggatcca ataaaaaaag ggagcactgt attc 34
<210> 71
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrC-F
<400> 71
ggcgagctca tagagcgagg acgctaacag 30
<210> 72
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrC-R
<400> 72
ggcggatcca agaaaaaagg cgacagatta ctc 33
<210> 73
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrB-F2
<400> 73
ggcggatccc acacaggagg agctcgagtc 30
<210> 74
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrB-R2
<400> 74
ggcgcatgca ataaaaaaag ggagcactgt attc 34
<210> 75
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrC-F2
<400> 75
ggcggatccc acacaggagg agctcataga g 31
<210> 76
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer csrC-R2
<400> 76
ggcgcatgca agaaaaaagg cgacagatta ctc 33
- 上一篇：一种医用注射器针头装配设备
- 下一篇：修饰的CPF1引导RNA