九種載體的多克隆位點(diǎn)及質(zhì)粒圖譜

..九種載體的多克隆位點(diǎn)及質(zhì)粒圖譜〔pLLP-OmpA、pLLP-STII、pMBP-P、pMBP-C、pTrc-CKS、pET-GST、pET-Trx、pET-His、pET-DsbA〕這九種載體的多克隆位點(diǎn)均為GGATCCGCAGAATTCAGCGCTAGCCAC〔TAA〕CATCATBamHIEcoRINheIHisHisHisCATCATCATTAAGCTTHisHisHisHindIII我們建議您擴(kuò)增基因5端選BamHI或BglII，3端選NheI或XbaI或SpeI，同時3端不要帶終止密碼子，注意與GGATCC〔GlySer〕表達(dá)框架正確，載體用NheI—BamHI雙酶切。BamHI和BglII為同裂酶，NheI、XbaI和SpeI為同裂酶。如您目的基因無法滿足上述要求，如同時含有BamHI和BglII，5端可選擇EcoRI，但須注意與表達(dá)框架GAATTC〔GluPhe〕對齊。如您目的基因同時含有XbaI、NheI和SpeI，3端可選擇EcoRI。如果您的目的基因已帶有終止密碼子，同樣可以進(jìn)展克隆表達(dá)，只是注意有些表達(dá)載體是在C端帶6個His。PCR引物設(shè)計(jì)時BamHI和EcoRI位點(diǎn)的5端保護(hù)堿基有2至3個GC即可，而XbaI、NheI和SpeI位點(diǎn)5端保護(hù)堿基須在5個左右才容易被酶切割?！惨弧硃LLp-OmpASize:7.5Kb多克隆位點(diǎn)：PlpplacoperatorRBSCGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGMetLysLysThrAlaIleAlaOmpAsignalATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGGATCCGCAIleAlaValAlaLeuAlaGlyPheAlaThrValAlaGlnAlaBamHIGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPlpplacoperatorRBS—ATG—OmpAsignal—MCS—基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagataacgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta載體其它局部序列見GenebankLocusASAK3121(plasmidpIN-III-ompA1)〔二〕pLLp-STIISize:7.5kb多克隆住點(diǎn)：PlpplacoperatorRBSGAAGGAGATATACATATGAAAAAGAACATCGCATTCCTCMetLysLysAsnIleAlaPheLeuSTIIsignalCTGGCATCTATGTTTGTTTTCTCTATCGCTACCAACGCTTACGCTGGATCCLeuAlaSerMetPheValPheSerIleAlaThrAsnAlaTyrAlaBammHIGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPlpplacoperatorATG—STIIsignalpeptide—MCS—基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta載體其它局部序列見GenebankLocusASAK3121(plasmidpIN-III-ompA1)STIIsignalpeptide見GenebankLocusHSTI_ECOLI(p22542)(三)pMBP-pVectorsize:6.7kbPtaclacoperator分泌型maltosebindingproteinAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGGATCCGCAGAAAsnTyrProArgBamHIEcoRITTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPtaclacoperator分泌型MBP—thrombincutsiteMCS基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證GccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII載體其它局部序列見pMal-p2Maltosebindingprotein(E.coli)編碼氨基酸序列見GenebankMBPMW=42KD(四)pMBP-cVectorsize:6.6kbPtaclacoperator胞maltosebindingproteinAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGGATCCGCAGAAAsnTyrProArgBamHIEcoRITTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPtaclacoperator胞MBPthrombincutsite—MCS基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證GccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII載體其它局部序列見pMal-c2Maltosebindingprotein(E.coli)編碼氨基酸序列見GenebankMBPMW=42KD〔五〕pET-GSTvectorsize:3.7kb多克隆位點(diǎn)：P3CcutsitePT7RBS—ATG—GST—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCCACCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIBamHIEcoRINheI6HisstopHindIIIPT7GSTP3ccutsite—MCS—基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證XbaItctagatcacacaggaaacagtattcatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggacagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII載體其它局部序列見pUC18(不含pUC18多克隆位點(diǎn))GlutathioneS-transferase編碼氨基酸序列如下〔含多克隆位點(diǎn)〕：MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQGPAGSAEFSASHHHHHH248AMINOACIDSMW28.7KD〔六〕pET-Trxvectorsize:3.3kb多克隆位點(diǎn)：P3CcutsitePT7RBS—TRX(his-patch—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATTAAGCTTEcoRINheIstopHindIIIBamHIEcoRINheIstopHindIIIPT7p3ccutsite—MCS—基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證XbaItctagaaataattttgtttaactttaagaaggagatatacatatgtctgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindIII載體其它局部序列見pUC18(不含pUC18多克隆位點(diǎn))Thioredoxinmut(His-patch)(E.coli)編碼氨基酸序列如下〔含多克隆位點(diǎn)〕：MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS133AMINOACIDSMW14KD3個His形成立體His-patch〔七〕pET-Hisvectorsize:2.9kb多克隆位點(diǎn)：thrombincutsitePT7RBS—ATGHisCTGGTGCCGCGCGGATCCGCAGAATTCAGCGCTLeuValProArgBamHIEcoRIAGCTAACATCATCATCATCATTAAGCTTNheIHindIIIBamHIEcoRINheIstopHindIIIPT7ATG-6HisThrombincutsite—MCS—基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證XbaItctagaaataattttgtttaactttaagaaggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindIII載體其它局部序列見pUC18(不含pUC18多克隆位點(diǎn))(八)PTRC-CKSvectorsize:5.2kb多克隆位點(diǎn)：P3CcutsitePTrcRBS—ATG—CKSmut—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINheI6His-tagHindIIIBamHIEcoRINheI6His-stopHindIIIPTrc—RBS—ATG—CKSmut—p3Ccutsite—MCS基因克隆表達(dá)功能區(qū)序列如下，經(jīng)DNA雙向測序驗(yàn)證XbaItctagaaataattttgtttaactttaagaaggagatatacatatgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggcaaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgtatgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgctgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgttttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII載體其它局部序列見pTrcHisB(不含pTrcHisB多克隆位點(diǎn))CKSmut(E.coliCTP:CMP-3-deoxy-D-manno-octulosonatecytidylyltransferaseorCMP-KDOsynthetase)編碼氨基酸序列如下(含多克隆位點(diǎn))：MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEKCAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMATLAVPIHNAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH268AminoAcidsMolecularWeight29KD〔九〕PET-DsbAvectorsize:3.6kb多克隆位點(diǎn)thrombincutsitePT7RBS—ATG-6HisDsbAmut—ATGTATCCGCGTGGAMetTyrProArgBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATCATTAAGCTT3EcoRINheIstopHindIIIBamHIEcoR