pET28a全序列

1、pET28a全序列Bpu l 102 l(80)Dra 111(5127)小钝3-5358)Xho 1(158) Not 1(166) Eag 1(166)Hind 111(173)Sal 1(179)Sac 1(190)EcoR l(192|BamH 1(198), Nhe 1(231)' Nde 1(238)NCO 1(296)Xba 1(335) zBgl 11(401) y SgrA 1(442)Sph 1(598)PVU 1(4426)Sgf 1(4426)Sma 1(4300)Cla 1(4117)Nni 1(4083)pET-28a(+)(5369bp)基(773,185

2、2Eco57 1(3772)zMlu 1(1123)、Bcl 1(1137)BstE 11(1304：、Apd 1(1334)BSSH 11(1534)EcoR V(1573)Hpa 1(1629)AlwN 1(3640)BssS 1(3397)BspLU 1 1 1224) z 6叩 1(3108)广、Bst l 107 1(2995) zTth I 1 1 1(2969)/ PshA 1(1968)、Bgl 1(2187) Fsp 1(2205)Psp5 11(2230)T7 promoter primer 069348-3pET upstream primer #69214-3-T7 D

3、romotGr,u 而U1' Promoter , Wc operator XbalrbsAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAA7AATTTTGTTTAACTTTAAGAAGGAGANeo IHi$*TagNde I Nhe I T7*Tag-ATACCATGGGCAGCAGCCATCATCAT2A-2A-CACAGCAGCGGCC7GGTGCCGCGCGGCAGCCATATGGC-AGCATGACTGGTGGACAGCAAetGlySerSerHisHisHIsHisHi s

4、hisSerSeGIyLeuValPoA？Gly5"H sMetAIaSerKetlhrGlyGlyGInGIn E8 thrombin BamH I EccR I Sac I _Sa/l HE III 磷 I Xho I HisTagATGGGTCGCGGATCCGAA ' : 3TCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28ol*)MetGlyArgGlySerGIuPheGluLeuArqArQGI nA IoCysGIyArgThrArgAloProProProP

5、roProLeuArgSerGIyCysEnd.GGTCGGGATCCGAATTCGAGCTCCGTCGACAAGCT-GCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAAZAAAGCCC pET-28b|*).GIyArgAspProAsnSer5er5erVo：AspLysLeuAIaAIaAIaLeuGIuHisHisH sHisHishisEnd.GGTCGGATCCGAATTCGAGCTCCGTCGACA/iGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACMAGCCC pET

6、-28c|*). GlyArgllaArglleArgaiQnroserThrSgfL&uArgProHlsSerSerTirThrThfThrThrThrGlu】SApLejLeuTHrLysPro.fipul 102 I T7 terminatorGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAACGGGTCTTGAGGGGTTTTTTGT7 terminator primer #69337-3pET-28a-c(+) cloning/expression regionpET28a|(十) sequ

7、ence landmarksT7 promoterT7 transcription start HisTag coding sequence T7eTag coding sequence Multiple cloning sites (BamH I - Xho I) HiseTag coding sequence T7 terminator lad coding sequence pBR322 origin Kan coding sequence fl origin370-386369270-287207-239158-203140-15726-72773-18523286 3995-4807

8、 4903-53581ATCCGGATAT AGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTA 51 GAGGCCCCAA GGGGTTATGC TAGTTATTGCTCAGCGGTGG101CTCAGCTTCCrTTCGGGCTTTGTTAGCAGCCGGATCTCAGCAGCAGCCAATGGTGGTGGT 151GGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGA 201TCCGCGACCCCACCAGTCATTGGCTGCCGC 251GCCGCTGCTGATTTGCTGTCGCTAGCCATAGCGGCAC

9、CAGTGATGATGATGATGATGGCTGCTGCCCATG 301GTATATCTCC TTCTTAAAGT TAAACAAAATTATTTCTAGA GGGGAATTGT 351 TATCCGCTCA CAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCG 401 AGATCTCGAT CCTCTACGCC GGACGCATCGTGGCCGGCATCACCGGCGCC TTGCTGGCGC GACATCACCG TCGGGCTCGC TCATGAGCGC GTGGGTATGG CGTGGCCGGG451 ACAGGTGCGG CTATATCGCCATGGGG

10、AAGA 501CACTTCGGGCTTGTTTCGGC551 TGGCAGGCCC GGACTGTTGGGCGCCATCTCCTTGCATGCA601CCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTG651CTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACC701ATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGA751GAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCG801CAGAGTATGCCG

11、GTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAG851GCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGC901GGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAAC951AGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCG 1001 TCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAAC TGGGTGCCAG 1051CGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCT

12、GTAAAGCGG 1101CGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGAT CATTAACTAT1151CCGCTGGATGCATTGCTGTGACCAGGATGCGAAGCTGCCTGCACTAATGT 1201TCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATC AACAGTATTA 1251 TTTTCTCCCA TGAAGACGGT ACGCGACTGGGCGTGGAGCATCTGGTCGCA1301 TTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTC 1351GGCGCGT

13、CTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAA1401 TTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCAT GTCCGGTTTT1451CAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCT1501 GGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT 1551CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACC1601 GAAGACAGCTCATGTTATATCCCGCCGTTAAC

14、CACCATCA AACAGGATTT1651TCGCCTGCTG GGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGG 1701 GCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACT GGTGAAAAGA1751AAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC1801CGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG GAAAGCGGGC 1851AGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGG1901A

15、TCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGC TCCTTCCGGT1951GGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATC 2001ATGCAACTCGTAGGACAGGT CTCTGGGTCA GGACCGCTTT CGACGATGAT CTTGCGGTAT GCACGCCCTC TCGTCACTGG AAACGTTTCG GGCCATTATC CGGCCCCACG ATCGTGCTCCTGGTTAGCAG CGATACGCGA AGCGACTGCTGCCGGCAGCGTTTTCGGCGA 2051 CGC

16、TGGAGCG CGGCCTGTCG2101 TCGGAATCTT GCTCAAGCCTTCCCGCCACC 2151 GCGAGAAGCA GCCGGCATGG 2201 GGTGCGCATGTGTCGTTGAGGACCCGGCTA GGCTGGCGGG 2251GTTGCCTTACAATGAATCAC GCGAACGTGA 2301GCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGT 2351CTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCT2401 GCACCATTATGTTCCGGATCTGCATCGCAGG

17、ATGCTGCTGGCTACCCTGT 2451GGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGAT2501 TTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAAC 2551GTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATC2601 CTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAA ATCCCCCTTA 2651 CACGGAGGCA TCAGTGACCA AACAGGAAAAAACCGCCCTTAACATGGCCC27

18、01 GCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTG 2751GACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGA2801 TGAGCTTTACCGCAGCTGCCTCGCGCGTTTTTGGCGGGTG GCCATGACCC CGATAGCGGA GCTTAACTAT AGCAGATTGT CACCATATAT ATACCGCACA GAGAAAATAC GCTCTTCCGC ACTGACTCGC CGTTCGGCTG TATCAGCTCACGGTGATGAC GGTGAAAACC 2851

19、TCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGAT 2901GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG2951 TCGGGGCGCA AGTCACGTAG GTGTATACTG 3001GCGGCATCAG ACTGAGAGTG 3051 GCGGTGTGAA GATGCGTAAG CGCATCAGGC 3101 TTCCTCGCTC TGCGCTCGGT 3151 CGGCGAGCGG CTCAAAGGCGGTAATACGGT TATCCACAGA 3201ATCAGGGGAT AGAACATGTG CA

20、GCAAAAGG TAAAAAGGCC CGTTTTTCCAAACGCAGGAA AGCAAAAGGC 3251 CCAGGAACCG GCGTTGCTGG TAGGCTCCGC 3301CCCCCTGACG AAATCGACGC GGTGGCGAAA CTATAAAGAT TCCCCCTGGA TGCGCTCTCC CTGCCGCTTA GTCCGCCTTT GAAGCGTGGCAGCATCACAA TCAAGTCAGA3351 CCCGACAGGA ACCAGGCGTTAGCTCCCTCG 3401 TGTTCCGACC CCGGATACCT3451 CTCCCTTCGG GCTTTCTC

21、ATAGCTCACGCTGTAGGTATCT 3501CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC3551CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC 3601AACCCGGTAAGACACGACTTATCGCCACTG CTGGTAACAG GCGAGGTATG TACAGAGTTC GGCCTAACTA AGAAGGACAG CTGCGCTCTG TTACCTTCGG GGTAGCTCTT ACAAACCACC GTGGTTTTTT CAGCAGATTA AAAAGGATCT C

22、TTTGATCTT TCTGACGCTC AAACTCACGT TGGTCATGAA GTCTGCTTAC3751 CTGAAGCCAGGCAGCAGCCA3651 GATTAGCAGATAGGCGGTGCTTGAAGTGGT 3701CGGCTACACT TATTTGGTATAAAAAGAGTTGATCCGGCAA 3801 GCTGGTAGCG TGTTTGCAAG3851 CGCGCAGAAACAAGAAGATCTTCTACGGGG 3901AGTGGAACGA TAAGGGATTT 3951 CAATAAAACT ATAAACAGTAATACAAGGGG TGTTATGAGC4001

23、CATATTCAAC GGGAAACGTC TTGCTCTAGGCCGCGATTAAATTCCAACAT 4051 GGATGCTGATTTATATGGGT ATAAATGGGC TCGCGATAAT GTCGGGCAAT4101 CAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTG4151TTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGAT 4201GGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGC4251ATTTTATCCGTACTCCTGATG

24、ATGCATGGTTACTCACCAC TGCGATCCCC4301 GGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAA4351TATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTG 4401TTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCG4451CAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGA 4501GCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTT4551 TGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTT CTCACTTGAT4601 AACCTTATT1TTGACGAGGGGGTTGTATTGGCAGACCGAT TGCCATCCTA TCGGTGAGTT TTACAGAAACGA