pET-30a大肠表达质粒

基本信息

启动子： T7/lac

复制子： ColE1 ori，F1 ori

终止子： T7 terminator

质粒分类： 大肠杆菌载体；PET系列表达质粒

质粒大小： 5422bp

质粒标签： N-6×His, N-S, N-Thrombin, N-EK, C-6×His

原核抗性： 卡那霉素Kan

克隆菌株： DH5α

培养条件： 37℃，有氧，LB

表达宿主： BL21(DE3)

诱导方式： IPTG或乳糖及其类似物

5'测序引物： T7：TAATACGACTCACTATAGGG

3'测序引物： T7-ter：TGCTAGTTATTGCTCAGCGG

二,质粒简介

The pET-30a-c(+) vectors carry an N-terminal His• Tag®/ thrombin/ S• Tag™/ enterokinase configuration plus an optional C-terminal His• Tag sequence. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer .

ORIGIN

1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa

61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt

121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt

181 cgacggagct cgaattcgga tccgatatca gccatggcct tgtcgtcgtc gtcggtaccc

241 agatctgggc tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatacca

301 gaaccgcgtg gcaccagacc agaagaatga tgatgatgat ggtgcatatg tatatctcct

361 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta

421 tagtgagtcg tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca

481 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca

541 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta

601 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc

661 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt

721 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt

781 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg

841 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac

901 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg

961 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc

1021 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct

1081 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa

1141 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac

1201 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg

1261 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac

1321 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta

1381 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc

1441 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt

1501 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc

1561 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt

1621 gcggacatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg

1681 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg

1741 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa

1801 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca

1861 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat

1921 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt

1981 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac

2041 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg

2101 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat

2161 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa

2221 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt

2281 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg

2341 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat

2401 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat

2461 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt

2521 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac

2581 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg

2641 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc

2701 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat

2761 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc

2821 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg

2881 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg

2941 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg

3001 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac

3061 tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc

3121 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact

3181 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac

3241 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa

3301 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg

3361 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa

3421 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc

3481 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac

3541 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac

3601 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg

3661 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt

3721 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga

3781 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct

3841 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga

3901 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg

3961 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct

4021 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct

4081 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat

4141 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag

4201 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga

4261 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct

4321 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa

4381 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg

4441 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag

4501 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat

4561 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat

4621 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta

4681 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc

4741 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat

4801 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc

4861 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg

4921 ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt gtaaacgtta atattttgtt

4981 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg

5041 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg

5101 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta

5161 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg

5221 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa

5281 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct

5341 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct

5401 acagggcgcg tcccattcgc ca