九种载体的多克隆位点及质粒图谱

..九种载体的多克隆位点及质粒图谱〔pLLP-OmpA、pLLP-STII、pMBP-P、pMBP-C、pTrc-CKS、pET-GST、pET-Trx、pET-His、pET-DsbA〕这九种载体的多克隆位点均为GGATCCGCAGAATTCAGCGCTAGCCAC〔TAA〕CATCATBamHIEcoRINheIHisHisHisCATCATCATTAAGCTTHisHisHisHindIII我们建议您扩增基因5端选BamHI或BglII，3端选NheI或XbaI或SpeI，同时3端不要带终止密码子，注意与GGATCC〔GlySer〕表达框架正确，载体用NheI—BamHI双酶切。BamHI和BglII为同裂酶，NheI、XbaI和SpeI为同裂酶。如您目的基因无法满足上述要求，如同时含有BamHI和BglII，5端可选择EcoRI，但须注意与表达框架GAATTC〔GluPhe〕对齐。如您目的基因同时含有XbaI、NheI和SpeI，3端可选择EcoRI。如果您的目的基因已带有终止密码子，同样可以进展克隆表达，只是注意有些表达载体是在C端带6个His。PCR引物设计时BamHI和EcoRI位点的5端保护碱基有2至3个GC即可，而XbaI、NheI和SpeI位点5端保护碱基须在5个左右才容易被酶切割。〔一〕pLLp-OmpASize:7.5Kb多克隆位点：PlpplacoperatorRBSCGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGMetLysLysThrAlaIleAlaOmpAsignalATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGGATCCGCAIleAlaValAlaLeuAlaGlyPheAlaThrValAlaGlnAlaBamHIGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPlpplacoperatorRBS—ATG—OmpAsignal—MCS—基因克隆表达功能区序列如下，经DNA双向测序验证ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagataacgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta载体其它局部序列见GenebankLocusASAK3121(plasmidpIN-III-ompA1)〔二〕pLLp-STIISize:7.5kb多克隆住点：PlpplacoperatorRBSGAAGGAGATATACATATGAAAAAGAACATCGCATTCCTCMetLysLysAsnIleAlaPheLeuSTIIsignalCTGGCATCTATGTTTGTTTTCTCTATCGCTACCAACGCTTACGCTGGATCCLeuAlaSerMetPheValPheSerIleAlaThrAsnAlaTyrAlaBammHIGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPlpplacoperatorATG—STIIsignalpeptide—MCS—基因克隆表达功能区序列如下，经DNA双向测序验证ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta载体其它局部序列见GenebankLocusASAK3121(plasmidpIN-III-ompA1)STIIsignalpeptide见GenebankLocusHSTI_ECOLI(p22542)(三)pMBP-pVectorsize:6.7kbPtaclacoperator分泌型maltosebindingproteinAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGGATCCGCAGAAAsnTyrProArgBamHIEcoRITTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPtaclacoperator分泌型MBP—thrombincutsiteMCS基因克隆表达功能区序列如下，经DNA双向测序验证GccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII载体其它局部序列见pMal-p2Maltosebindingprotein(E.coli)编码氨基酸序列见GenebankMBPMW=42KD(四)pMBP-cVectorsize:6.6kbPtaclacoperator胞maltosebindingproteinAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGGATCCGCAGAAAsnTyrProArgBamHIEcoRITTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPtaclacoperator胞MBPthrombincutsite—MCS基因克隆表达功能区序列如下，经DNA双向测序验证GccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII载体其它局部序列见pMal-c2Maltosebindingprotein(E.coli)编码氨基酸序列见GenebankMBPMW=42KD〔五〕pET-GSTvectorsize:3.7kb多克隆位点：P3CcutsitePT7RBS—ATG—GST—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCCACCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIBamHIEcoRINheI6HisstopHindIIIPT7GSTP3ccutsite—MCS—基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagatcacacaggaaacagtattcatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggacagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII载体其它局部序列见pUC18(不含pUC18多克隆位点)GlutathioneS-transferase编码氨基酸序列如下〔含多克隆位点〕：MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQGPAGSAEFSASHHHHHH248AMINOACIDSMW28.7KD〔六〕pET-Trxvectorsize:3.3kb多克隆位点：P3CcutsitePT7RBS—TRX(his-patch—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATTAAGCTTEcoRINheIstopHindIIIBamHIEcoRINheIstopHindIIIPT7p3ccutsite—MCS—基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagaaataattttgtttaactttaagaaggagatatacatatgtctgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindIII载体其它局部序列见pUC18(不含pUC18多克隆位点)Thioredoxinmut(His-patch)(E.coli)编码氨基酸序列如下〔含多克隆位点〕：MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS133AMINOACIDSMW14KD3个His形成立体His-patch〔七〕pET-Hisvectorsize:2.9kb多克隆位点：thrombincutsitePT7RBS—ATGHisCTGGTGCCGCGCGGATCCGCAGAATTCAGCGCTLeuValProArgBamHIEcoRIAGCTAACATCATCATCATCATTAAGCTTNheIHindIIIBamHIEcoRINheIstopHindIIIPT7ATG-6HisThrombincutsite—MCS—基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagaaataattttgtttaactttaagaaggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindIII载体其它局部序列见pUC18(不含pUC18多克隆位点)(八)PTRC-CKSvectorsize:5.2kb多克隆位点：P3CcutsitePTrcRBS—ATG—CKSmut—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPTrc—RBS—ATG—CKSmut—p3Ccutsite—MCS基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagaaataattttgtttaactttaagaaggagatatacatatgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggcaaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgtatgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgctgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgttttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII载体其它局部序列见pTrcHisB(不含pTrcHisB多克隆位点)CKSmut(E.coliCTP:CMP-3-deoxy-D-manno-octulosonatecytidylyltransferaseorCMP-KDOsynthetase)编码氨基酸序列如下(含多克隆位点)：MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEKCAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMATLAVPIHNAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH268AminoAcidsMolecularWeight29KD〔九〕PET-DsbAvectorsize:3.6kb多克隆位点thrombincutsitePT7RBS—ATG-6HisDsbAmut—ATGTATCCGCGTGGAMetTyrProArgBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATCATTAAGCTT3EcoRINheIstopHindIIIBamHIEcoR