九種載體的多克隆位點(diǎn)及質(zhì)粒圖譜

1、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)CATCATBamHIEcoRINhelHisHisHisCATCATCATTAAGCTTHisHisHisHindlll我們建議您擴(kuò)增基因5瑞選BamHI或Bglll,3湍選Nhel或Xbal或Spel,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NhelBamHI雙酶切。BamHI和

2、Bglll為同裂酶，Nhel、Xbal和Spel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Bglll,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Xbal、Nhel和Spel,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)行克隆表達(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Xbal、Nhel和Spel位點(diǎn)5,端保護(hù)堿基須在5個左右才容易被酶切割。(一)pLLp-OmpASize:7.5Kb多克隆位點(diǎn)：Plpplacopera

3、torRBSICGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGMetLysLysThrAlaIleAlaOmpAsignalATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGGATCCGCAIleAlaValAlaLeuAlaGlyPheAlaThrValAlaGlnAlaBamHIGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINhel6His-tagHindlllBamHIEcoRINheI6His-stopHindIIIOri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ctcacttta

4、tctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagata

5、acgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtt

6、taccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta載體其它部分序列見GenebankLocusASAK3121(plasmidpIN-III-ompAI)(二)pLLp-STIISize:7.5kblacoperatorRBSPlpp多克隆住點(diǎn):GAAGGAGATATACATATGAAAAAGAACATCGCATTCCTCMetLysLysAsn

7、IleAlaPheLeuSTIIsignalCTGGCATCTATGTTTGTTTTCTCTATCGCTACCAACGCTTACGCTGGATCCLeuAlaSerMetPheValPheSerIleAlaThrAsnAlaTyrAlaBammHIGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagHindlllBamHIEcoRINhel6His-stopHindlllOri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ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaattttttta

8、tctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacat

9、cgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgta

10、acatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta載體其它部分序列見GenebankLocusASAK3121(plasmidpIN-III-ompAI)STIIsignalpeptide見GenebankLocusHSTI_ECOLI(p22542)(三)pMBP-pVectorsize:6.7kbPtaclacoperator分泌型maltosebindingprotein|AACAA

11、CAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGgAtCCGCAGAAHindlllTTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagBamHIEcoRINhel6His-stopHindlllOri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Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagc

12、ggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll載體其它部分序列見pMal-p2Maltosebindingprotein(E.coli)編碼氨基酸序列見GenebankMBPMW=42KD(四)pMBP-cVectorsize:6.6kbPtaclacoperator.1的內(nèi)maltosebindi

13、ngprotenAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGgAtCCGCAGAAHindlllTTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagOri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Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcga

14、tgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll載體其它部分序列見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)：P3CcutsitePT7|RBS|一|ATGGST|CTCGAAGTTCTGTT

15、TCAG%GTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHlTCCGCAGAATTCAGCGCTAGCCACCATCATCATCATTAAGCTT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XbaItctagatcacacaggaaacagtattatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttccc

16、aatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttc

17、atgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggacagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll載

18、體其它部分序列見pUC18（不含pUC18多克隆位點(diǎn)）GlutathioneS-transferas卻碼氨基酸序列如下（含多克隆位點(diǎn)）：MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQ

19、GPAGSAEFSASHHHHHH248AMINOACIDSMW28.7KD（六）pET-Trxvectorsize:3.3kb多克隆位點(diǎn)：P3CcutsitePT7|RBS|TRX（his-patCCTCGAAGTTCTGTTTCAG*GGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATTAAGCTTEcoRINhelstopHindlllQri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XbaltctagaaataattttgtttaactttaagaaggagatatacaOtgtc

20、tgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaaga

21、gttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindlll載體其它部分序列見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))MSDKllHLTDDSFDTDVLKADGAlLVDFWAHWCGPCKMlAPlLDElADEYQGKLTVAKLNlDHNPGTAPKYGlRGlPTLLLFKNGEVAATKVGALS

22、KGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS133AMlNOAClDSMW14KD3個His形成立體His-patch（七）pET-Hisvectorsize:2.9kb多克隆位點(diǎn)：thrombincutsitePT7RBSATGHisCTGGTGCCGCGC*GGATCCGCAGAATTCAGCGCTLeuValProArgBamHIEcoRIAGCTAACATCATCATCATCATTAAGCTTNhelHindlllBamHIEcoRINhelstopHindlllQri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Xbaltctagaaataattttg

23、tttaactttaagaaggagatatacatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindlll載體其它部分序列見pUC18（不含pUC18多克隆位點(diǎn)）（八）PTRC-CKSvectorsize:5.2kb多克隆位點(diǎn)：P3CcutsitePTrc|RBS|AtGCKSmuHCTCGAAGTTCTGTTTCAG%GTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGC

24、CACCATCATCATCATCATTAAGCTTBamHIEcoRINhel6His-stopHindlllPTrcRBSATGCKSmutp3ccutsiteMCSOri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XbaItctagaaataattttgtttaactttaagaaggagatatacaihtgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggc

25、aaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgtatgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgc

26、tgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgttttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagt

27、tctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII載體其它部分序列見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)）：MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEK

28、CAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMolecularWeight29KDMATLAVPIHNAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH268AminoAcids（九）PET-DsbAvectorsize:3.6kbthrombincutsite多克隆位點(diǎn)PT7RBS|ATG-6HisDsbAmut卜ATGTATCCGCGT%GAMetTyrProArgBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATCATTAAGCTT3EcoRINhelstopHindlllQri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）XbaITctagaaataattttgtttaactttaagaaggagatatacatgggcagctctcatcatcatcatcatcactctagcggtgctcagtacgaagatggtaaaca