pTWIN1

pTWIN1载体基本信息

pTWIN1载体质粒图谱和多克隆位点信息

pTWIN1载体简介

pTWIN1 is an E. coli plasmid cloning vector designed for recombinant protein expression, labeling, and cyclization using the IMPACT-TWIN Kit (NEB #E6901) (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTWIN1 also contains an M13 origin of replication.

The multiple cloning site (MCS) is positioned to allow translational fusion of an intein tag to the N-terminus, C-terminus, or both, of the cloned target protein. The mini-inteins encoded by pTWIN1, the Ssp DnaB intein and the Mxe GyrA intein, cleave the peptide bond at their C- and N-termini, respectively (1-4). The chitin binding domain (CBD) from B. circulans, fused to each intein, facilitates purification of the intein-target protein precursor.

Transcription of the intein tags is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g., NEB #C2566, #C2833 or BL21(DE3)] for expression. Basal expression from the T7 promoter is minimized by the binding of the Lac repressor, encoded by the lacI gene, to the lac operator immediately downstream of the T7 promoter (5). Translation of the intein tags utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).

pTWIN1载体序列

ORIGIN

    1 AACTACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT

   61 CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA

  121 ATATTGAAAA AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT

  181 TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC

  241 TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT

  301 CCTTGAGAGT TTTCGCCCCG AAGAACGTTC TCCAATGATG AGCACTTTTA AAGTTCTGCT

  361 ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA

  421 CTATTCTCAG AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG

  481 CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA

  541 CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG

  601 GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA

  661 CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG

  721 CGAACTACTT ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT

  781 TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG

  841 AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC

  901 CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA

  961 GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC

 1021 ATATATACTT TAGATTGATT TACCCCGGTT GATAATCAGA AAAGCCCCAA AAACAGGAAG

 1081 ATTGTATAAG CAAATATTTA AATTGTAAAC GTTAATATTT TGTTAAAATT CGCGTTAAAT

 1141 TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA

 1201 TCAAAAGAAT AGCCCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA

 1261 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA

 1321 CTACGTGAAC CATCACCCAA ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT

 1381 CGGAACCCTA AAGGGAGCCC CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG

 1441 AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC

 1501 ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTAAAAG

 1561 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC

 1621 GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT

 1681 TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT

 1741 GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT

 1801 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC

 1861 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA

 1921 GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG

 1981 CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG

 2041 ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG

 2101 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA

 2161 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT

 2221 GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG

 2281 GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC

 2341 TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC

 2401 CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC TATGGTGCAC TCTCAGTACA ATCTGCTCTG

 2461 ATGCCGCATA GTTAAGCCAG TATACACTCC GCTATCGCTA CGTGACTGGG TCATGGCTGC

 2521 GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC TCCCGGCATC

 2581 CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT TTTCACCGTC

 2641 ATCACCGAAA CGCGCGAGGC AGCTGCGGTA AAGCTCATCA GCGTGGTCGT GCAGCGATTC

 2701 ACAGATGTCT GCCTGTTCAT CCGCGTCCAG CTCGTTGAGT TTCTCCAGAA GCGTTAATGT

 2761 CTGGCTTCTG ATAAAGCGGG CCATGTTAAG GGCGGTTTTT TCCTGTTTGG TCACTGATGC

 2821 CTCCGTGTAA GGGGGATTTC TGTTCATGGG GGTAATGATA CCGATGAAAC GAGAGAGGAT

 2881 GCTCACGATA CGGGTTACTG ATGATGAACA TGCCCGGTTA CTGGAACGTT GTGAGGGTAA

 2941 ACAACTGGCG GTATGGATGC GGCGGGACCA GAGAAAAATC ACTCAGGGTC AATGCCAGCC

 3001 GAACGCCAGC AAGACGTAGC CCAGCGCGTC GGCCGCCATG CCGGCGATAA TGGCCTGCTT

 3061 CTCGCCGAAA CGTTTGGTGG CGGGACCAGT GACGAAGGCT TGAGCGAGGG CGTGCAAGAT

 3121 TCCGAATACC GCAAGCGACA GGCCGATCAT CGTCGCGCTC CAGCGAAAGC GGTCCTCGCC

 3181 GAAAATGACC CAGAGCGCTG CCGGCACCTG TCCTACGAGT TGCATGATAA AGAAGACAGT

 3241 CATAAGTGCG GCGACGATAG TCATGCCCCG CGCCCACCGG AAGGAGCTGA CTGGGTTGAA

 3301 GGCTCTCAAG GGCATCGGTC GAGATCCCGG TGCCTAATGA GTGAGCTAAC TTACATTAAT

 3361 TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG

 3421 AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCCAGGGTG GTTTTTCTTT

 3481 TCACCAGTGA GACGGGCAAC AGCTGATTGC CCTTCACCGC CTGGCCCTGA GAGAGTTGCA

 3541 GCAAGCGGTC CACGCTGGTT TGCCCCAGCA GGCGAAAATC CTGTTTGATG GTGGTTAACG

 3601 GCGGGATATA ACATGAGCTG TCTTCGGTAT CGTCGTATCC CACTACCGAG ATATCCGCAC

 3661 CAACGCGCAG CCCGGACTCG GTAATGGCGC GCATTGCGCC CAGCGCCATC TGATCGTTGG

 3721 CAACCAGCAT CGCAGTGGGA ACGATGCCCT CATTCAGCAT TTGCATGGTT TGTTGAAAAC

 3781 CGGACATGGC ACTCCAGTCG CCTTCCCGTT CCGCTATCGG CTGAATTTGA TTGCGAGTGA

 3841 GATATTTATG CCAGCCAGCC AGACGCAGAC GCGCCGAGAC AGAACTTAAT GGGCCCGCTA

 3901 ACAGCGCGAT TTGCTGGTGA CCCAATGCGA CCAGATGCTC CACGCCCAGT CGCGTACCGT

 3961 CTTCATGGGA GAAAATAATA CTGTTGATGG GTGTCTGGTC AGAGACATCA AGAAATAACG

 4021 CCGGAACATT AGTGCAGGCA GCTTCCACAG CAATGGCATC CTGGTCATCC AGCGGATAGT

 4081 TAATGATCAG CCCACTGACG CGTTGCGCGA GAAGATTGTG CACCGCCGCT TTACAGGCTT

 4141 CGACGCCGCT TCGTTCTACC ATCGACACCA CCACGCTGGC ACCCAGTTGA TCGGCGCGAG

 4201 ATTTAATCGC CGCGACAATT TGCGACGGCG CGTGCAGGGC CAGACTGGAG GTGGCAACGC

 4261 CAATCAGCAA CGACTGTTTG CCCGCCAGTT GTTGTGCCAC GCGGTTGGGA ATGTAATTCA

 4321 GCTCCGCCAT CGCCGCTTCC ACTTTTTCCC GCGTTTTCGC AGAAACGTGG CTGGCCTGGT

 4381 TCACCACGCG GGAAACGGTC TGATAAGAGA CACCGGCATA CTCTGCGACA TCGTATAACG

 4441 TTACTGGTTT CACATTCACC ACCCTGAATT GACTCTCTTC CGGGCGCTAT CATGCCATAC

 4501 CGCGAAAGGT TTTGCGCCAT TCGATGGTGT CCGGGATCTC GACGCTCTCC CTTATGCGAC

 4561 TCCTGCATTA GGAAGCAGCC CAGTAGTAGG TTGAGGCCGT TGAGCACCGC CGCCGCAAGG

 4621 AATGGTGCAT GCCGGCATGC CGCCCTTTCG TCTTCAAGAA TTAATTCCCA ATTCCCCAGG

 4681 CATCAAATAA AACGAAAGGC TCAGTCGAAA GACTGGGCCT TTCGTTTTAT CTGTTGTTTG

 4741 TCGGTGAACG CTCTCCTGAG TAGGACAAAT CCGCCGGGAG CGGATTTGAA CGTTGCGAAG

 4801 CAACGGCCCG GAGGGTGGCG GGCAGGACGC CCGCCATAAA CTGCCAGGAA TTAATTCCCC

 4861 AGGCATCAAA TAAAACGAAA GGCTCAGTCG AAAGACTGGG CCTTTCGTTT TATCTGTTGT

 4921 TTGTCGGTGA ACGCTCTCCT GAGTAGGACA AATCCGCCGG GAGCGGATTT GAACGTTGCG

 4981 AAGCAACGGC CCGGAGGGTG GCGGGCAGGA CGCCCGCCAT AAACTGCCAG GAATTAATTC

 5041 CCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT

 5101 TGTTTGTCGG TGAACGCTCT CCTGAGTAGG ACAAATCCGC CGGGAGCGGA TTTGAACGTT

 5161 GCGAAGCAAC GGCCCGGAGG GTGGCGGGCA GGACGCCCGC CATAAACTGC CAGGAATTAA

 5221 TTCCCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC

 5281 TGTTGTTTGT CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC

 5341 GTTGCGAAGC AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGAAT

 5401 TAATTCCCCA GGCATCAAAT AAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT

 5461 ATCTGTTGTT TGTCGGTGAA CGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG

 5521 AACGTTGCGA AGCAACGGCC CGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG

 5581 AATTGGGGAT CGGAATTAAT TCCCGGTTTA AACCGGGGAT CTCGATCCCG CGAAATTAAT

 5641 ACGACTCACT ATAGGGGAAT TGTGAGCGGA TAACAATTCC CCTCTAGAAA TAATTTTGTT

 5701 TAACTTTAAG AAGGAGATAT ACATATGAAA ATCGAAGAAG GTAAACTGAC AAATCCTGGT

 5761 GTATCCGCTT GGCAGGTCAA CACAGCTTAT ACTGCGGGAC AATTGGTCAC ATATAACGGC

 5821 AAGACGTATA AATGTTTGCA GCCCCACACC TCCTTGGCAG GATGGGAACC ATCCAACGTT

 5881 CCTGCCTTGT GGCAGCTTCA AAACAACGGT AACAACGGTC TCGAACTGCG CGAGTCCGGA

 5941 GCTATCTCTG GCGATAGTCT GATCAGCCTG GCTAGCACAG GAAAAAGAGT TTCTATTAAA

 6001 GATTTGTTAG ATGAAAAAGA TTTTGAAATA TGGGCAATTA ATGAACAGAC GATGAAGCTA

 6061 GAATCAGCTA AAGTTAGTCG TGTATTTTGT ACTGGCAAAA AGCTAGTTTA TATTCTAAAA

 6121 ACTCGACTAG GTAGAACTAT CAAGGCAACA GCAAATCATA GATTTTTAAC TATTGATGGT

 6181 TGGAAAAGAT TAGATGAGCT ATCTTTAAAA GAGCATATTG CTCTACCCCG TAAACTAGAA

 6241 AGCTCCTCTT TACAATTGTC ACCAGAAATA GAAAAGTTGT CTCAGAGTGA TATTTACTGG

 6301 GACTCCATCG TTTCTATTAC GGAGACTGGA GTCGAAGAGG TTTTTGATTT GACTGTGCCA

 6361 GGACCACATA ACTTTGTCGC GAATGACATC ATTGTACACA ACGGAAGAGC CATGGGCGGC

 6421 CGCGAATTCC TCGAGGGCTC TTCCTGCATC ACGGGAGATG CACTAGTTGC CCTACCCGAG

 6481 GGCGAGTCGG TACGCATCGC CGACATCGTG CCGGGTGCGC GGCCCAACAG TGACAACGCC

 6541 ATCGACCTGA AAGTCCTTGA CCGGCATGGC AATCCCGTGC TCGCCGACCG GCTGTTCCAC

 6601 TCCGGCGAGC ATCCGGTGTA CACGGTGCGT ACGGTCGAAG GTCTGCGTGT GACGGGCACC

 6661 GCGAACCACC CGTTGTTGTG TTTGGTCGAC GTCGCCGGGG TGCCGACCCT GCTGTGGAAG

 6721 CTGATCGACG AAATCAAGCC GGGCGATTAC GCGGTGATTC AACGCAGCGC ATTCAGCGTC

 6781 GACTGTGCAG GTTTTGCCCG CGGAAAACCC GAATTTGCGC CCACAACCTA CACAGTCGGC

 6841 GTCCCTGGAC TGGTGCGTTT CTTGGAAGCA CACCACCGAG ACCCGGACGC CCAAGCTATC

 6901 GCCGACGAGC TGACCGACGG GCGGTTCTAC TACGCGAAAG TCGCCAGTGT CACCGACGCC

 6961 GGCGTGCAGC CGGTGTATAG CCTTCGTGTC GACACGGCAG ACCACGCGTT TATCACGAAC

 7021 GGGTTCGTCA GCCACGCTAC TGGCCTCACC GGTCTGAACT CAGGCCTCAC GACAAATCCT

 7081 GGTGTATCCG CTTGGCAGGT CAACACAGCT TATACTGCGG GACAATTGGT CACATATAAC

 7141 GGCAAGACGT ATAAATGTTT GCAGCCCCAC ACCTCCTTGG CAGGATGGGA ACCATCCAAC

 7201 GTTCCTGCCT TGTGGCAGCT TCAATGACTG CAGGAAGGGG ATCCGGCTGC TAACAAAGCC

 7261 CGAAAGGAAG CTGAGTTGGC TGCTGCCACC GCTGAGCAAT AACTAGCATA ACCCCTTGGG

 7321 GCCTCTAAAC GGGTCTTGAG GGGTTTTTTG CTGAAAGGAG GAACTATATC CGGAT

//

其他大肠杆菌表达载体：