pET-52b质粒

一,质粒描述：
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： 5227bp

质粒标签： N-Strep-Tag II，N-HRV 3C，C-Thrombin，C-10*His

原核抗性： 氨苄青霉素Amp

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

质粒简介

The pET-52b(+) vector carries an N-terminal Strep•Tag® II coding sequence (1) followed by a recognition site for the human rhinovirus (HRV) 3C protease. This protease is highly specific for cleavage of the sequence LEVLFQ↓GP (2), and is active at low temperatures (3). The multiple cloning region is followed by an optional thrombin recognition site and a C-terminal His•Tag® coding sequence. The presence of two “gentle elution" tags at both the N-terminus and C-terminus is ideal for dual purification strategies designed to isolate full-length fusion proteins (4). Unique restriction 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 circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below the circle map. The f1 origin is oriented so that infection with the helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand.

ORIGIN

1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg

61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc

121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg

181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc

241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt

301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc

361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta

421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat

481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt

541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt

601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc

661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg

721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc

781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg

841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca

901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga

961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct

1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg

1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca

1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata

1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct

1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact

1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa

1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc

1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg

1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac

1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag

1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg

1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca

1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga

1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca

1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc

1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca

1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa

2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc

2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc

2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg

2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat

2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca

2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt

2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac

2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg

2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg

2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg

2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg

2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga

2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg

2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa

2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt

2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt

3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct

3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac

3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag

3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc

3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc

3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc

3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga

3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt

3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt

3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg

3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc

3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat

3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt

3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc

3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc

3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag

3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt

4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat

4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag

4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac

4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg

4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg

4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt

4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc

4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact

4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg

4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga

4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc

4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt

4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg

4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc

4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag

4921 aaataatttt gtttaacttt aagaaggaga tataccatgg caagctggag ccacccgcag

4981 ttcgaaaagg gtgcacttga agtcctcttt cagggacccg ggtaccagga tcctgtacaa

5041 gtcgacgcgg ccgcagagct cgctctggtg ccacgcggta gttccgctca tcaccaccat

5101 catcaccatc accaccacta attaacctag gctgctgcca ccgctgagca ataactagca

5161 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata

5221 tccggat