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).

0001 AACTACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT 
0061 CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA 
0121 ATATTGAAAA AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT 
0181 TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC 
0241 TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT 
0301 CCTTGAGAGT TTTCGCCCCG AAGAACGTTC TCCAATGATG AGCACTTTTA AAGTTCTGCT 
0361 ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA 
0421 CTATTCTCAG AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG 
0481 CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA 
0541 CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG 
0601 GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA 
0661 CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG 
0721 CGAACTACTT ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT 
0781 TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG 
0841 AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC 
0901 CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA 
0961 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