pTWIN1
At a Glance
pTWIN1, Image provided by NEB.
<protect>
| Type |
Cloning & Expression |
|---|---|
| Origin of Replication |
pMB1 |
| Copy # |
~20 per cell |
| Markers |
Ampicillin |
| Link to Sequence | |
| Features |
rop |
| Accession Number |
|
| edit table |
</protect>
Notes
pTWIN1 is vector for expression of recombinant proteins within E. coli. The vector possesses a MCS between the Ssp DnaB and Mxe GyrA inteins that constitute the ORF. This MCS allows for insertion of a selected protein into the ORF. Utilization of the CBDs that are located at each end of the ORF allow for protein isolation. Fused gene expression is controlled by the T7 promoter, and is regulated by the lacI gene located immediately downstream of the T7 promoter.
History
The Ssp DnaB and Mxe GyrA mini-inteins are derived from the Synechocystis sp and Mycobacterium xenopi respectively. The chitin-binding domain (CBD) is derived from Bacillus circulans.
Sources
References
See Help:References for how to manage references in EcoliWiki.
- ↑ 1.0 1.1 1.2 Evans, TC Jr et al. (1999) The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins. J. Biol. Chem. 274 18359-63 PubMed
- ↑ 2.0 2.1 Evans, TC Jr et al. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7 2256-64 PubMed
- ↑ 3.0 3.1 Mathys, S et al. (1999) Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene 231 1-13 PubMed
- ↑ Chong, S et al. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192 271-81 PubMed
- ↑ Wu, H et al. (1998) Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein. Biochim. Biophys. Acta 1387 422-32 PubMed
- ↑ Telenti, A et al. (1997) The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein. J. Bacteriol. 179 6378-82 PubMed
Sequence
1 AACTACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT
51 GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA
101 CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA TGAGTATTCA
151 ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG
201 TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG
251 TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT
301 CCTTGAGAGT TTTCGCCCCG AAGAACGTTC TCCAATGATG AGCACTTTTA
351 AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG
401 CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG TTGAGTACTC
451 ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT
501 GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG
551 ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG
601 GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA
651 TACCAAACGA CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG
701 TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT CCCGGCAACA
751 ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT
801 CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG
851 CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC
901 CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC
951 GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA
1001 CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT TACCCCGGTT
1051 GATAATCAGA AAAGCCCCAA AAACAGGAAG ATTGTATAAG CAAATATTTA
1101 AATTGTAAAC GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA
1151 TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA
1201 TCAAAAGAAT AGCCCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA
1251 GAGTCCACTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG
1301 TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCAA ATCAAGTTTT
1351 TTGGGGTCGA GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC
1401 CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG AGAAAGGAAG
1451 GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC
1501 ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG
1551 CGCGTAAAAG GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA
1601 ATCCCTTAAC GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA
1651 GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT
1701 TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA
1751 GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT
1801 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA
1851 ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG
1901 GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG
1951 ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA
2001 CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG
2051 CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG
2101 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC
2151 CAGGGGGAAA CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC
2201 TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG
2251 GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC
2301 CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC
2351 CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC
2401 CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC TATGGTGCAC TCTCAGTACA
2451 ATCTGCTCTG ATGCCGCATA GTTAAGCCAG TATACACTCC GCTATCGCTA
2501 CGTGACTGGG TCATGGCTGC GCCCCGACAC CCGCCAACAC CCGCTGACGC
2551 GCCCTGACGG GCTTGTCTGC TCCCGGCATC CGCTTACAGA CAAGCTGTGA
2601 CCGTCTCCGG GAGCTGCATG TGTCAGAGGT TTTCACCGTC ATCACCGAAA
2651 CGCGCGAGGC AGCTGCGGTA AAGCTCATCA GCGTGGTCGT GCAGCGATTC
2701 ACAGATGTCT GCCTGTTCAT CCGCGTCCAG CTCGTTGAGT TTCTCCAGAA
2751 GCGTTAATGT CTGGCTTCTG ATAAAGCGGG CCATGTTAAG GGCGGTTTTT
2801 TCCTGTTTGG TCACTGATGC CTCCGTGTAA GGGGGATTTC TGTTCATGGG
2851 GGTAATGATA CCGATGAAAC GAGAGAGGAT GCTCACGATA CGGGTTACTG
2901 ATGATGAACA TGCCCGGTTA CTGGAACGTT GTGAGGGTAA ACAACTGGCG
2951 GTATGGATGC GGCGGGACCA GAGAAAAATC ACTCAGGGTC AATGCCAGCC
3001 GAACGCCAGC AAGACGTAGC CCAGCGCGTC GGCCGCCATG CCGGCGATAA
3051 TGGCCTGCTT CTCGCCGAAA CGTTTGGTGG CGGGACCAGT GACGAAGGCT
3101 TGAGCGAGGG CGTGCAAGAT TCCGAATACC GCAAGCGACA GGCCGATCAT
3151 CGTCGCGCTC CAGCGAAAGC GGTCCTCGCC GAAAATGACC CAGAGCGCTG
3201 CCGGCACCTG TCCTACGAGT TGCATGATAA AGAAGACAGT CATAAGTGCG
3251 GCGACGATAG TCATGCCCCG CGCCCACCGG AAGGAGCTGA CTGGGTTGAA
3301 GGCTCTCAAG GGCATCGGTC GAGATCCCGG TGCCTAATGA GTGAGCTAAC
3351 TTACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG
3401 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT
3451 GCGTATTGGG CGCCAGGGTG GTTTTTCTTT TCACCAGTGA GACGGGCAAC
3501 AGCTGATTGC CCTTCACCGC CTGGCCCTGA GAGAGTTGCA GCAAGCGGTC
3551 CACGCTGGTT TGCCCCAGCA GGCGAAAATC CTGTTTGATG GTGGTTAACG
3601 GCGGGATATA ACATGAGCTG TCTTCGGTAT CGTCGTATCC CACTACCGAG
3651 ATATCCGCAC CAACGCGCAG CCCGGACTCG GTAATGGCGC GCATTGCGCC
3701 CAGCGCCATC TGATCGTTGG CAACCAGCAT CGCAGTGGGA ACGATGCCCT
3751 CATTCAGCAT TTGCATGGTT TGTTGAAAAC CGGACATGGC ACTCCAGTCG
3801 CCTTCCCGTT CCGCTATCGG CTGAATTTGA TTGCGAGTGA GATATTTATG
3851 CCAGCCAGCC AGACGCAGAC GCGCCGAGAC AGAACTTAAT GGGCCCGCTA
3901 ACAGCGCGAT TTGCTGGTGA CCCAATGCGA CCAGATGCTC CACGCCCAGT
3951 CGCGTACCGT CTTCATGGGA GAAAATAATA CTGTTGATGG GTGTCTGGTC
4001 AGAGACATCA AGAAATAACG CCGGAACATT AGTGCAGGCA GCTTCCACAG
4051 CAATGGCATC CTGGTCATCC AGCGGATAGT TAATGATCAG CCCACTGACG
4101 CGTTGCGCGA GAAGATTGTG CACCGCCGCT TTACAGGCTT CGACGCCGCT
4151 TCGTTCTACC ATCGACACCA CCACGCTGGC ACCCAGTTGA TCGGCGCGAG
4201 ATTTAATCGC CGCGACAATT TGCGACGGCG CGTGCAGGGC CAGACTGGAG
4251 GTGGCAACGC CAATCAGCAA CGACTGTTTG CCCGCCAGTT GTTGTGCCAC
4301 GCGGTTGGGA ATGTAATTCA GCTCCGCCAT CGCCGCTTCC ACTTTTTCCC
4351 GCGTTTTCGC AGAAACGTGG CTGGCCTGGT TCACCACGCG GGAAACGGTC
4401 TGATAAGAGA CACCGGCATA CTCTGCGACA TCGTATAACG TTACTGGTTT
4451 CACATTCACC ACCCTGAATT GACTCTCTTC CGGGCGCTAT CATGCCATAC
4501 CGCGAAAGGT TTTGCGCCAT TCGATGGTGT CCGGGATCTC GACGCTCTCC
4551 CTTATGCGAC TCCTGCATTA GGAAGCAGCC CAGTAGTAGG TTGAGGCCGT
4601 TGAGCACCGC CGCCGCAAGG AATGGTGCAT GCCGGCATGC CGCCCTTTCG
4651 TCTTCAAGAA TTAATTCCCA ATTCCCCAGG CATCAAATAA AACGAAAGGC
4701 TCAGTCGAAA GACTGGGCCT TTCGTTTTAT CTGTTGTTTG TCGGTGAACG
4751 CTCTCCTGAG TAGGACAAAT CCGCCGGGAG CGGATTTGAA CGTTGCGAAG
4801 CAACGGCCCG GAGGGTGGCG GGCAGGACGC CCGCCATAAA CTGCCAGGAA
4851 TTAATTCCCC AGGCATCAAA TAAAACGAAA GGCTCAGTCG AAAGACTGGG
4901 CCTTTCGTTT TATCTGTTGT TTGTCGGTGA ACGCTCTCCT GAGTAGGACA
4951 AATCCGCCGG GAGCGGATTT GAACGTTGCG AAGCAACGGC CCGGAGGGTG
5001 GCGGGCAGGA CGCCCGCCAT AAACTGCCAG GAATTAATTC CCCAGGCATC
5051 AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT
5101 TGTTTGTCGG TGAACGCTCT CCTGAGTAGG ACAAATCCGC CGGGAGCGGA
5151 TTTGAACGTT GCGAAGCAAC GGCCCGGAGG GTGGCGGGCA GGACGCCCGC
5201 CATAAACTGC CAGGAATTAA TTCCCCAGGC ATCAAATAAA ACGAAAGGCT
5251 CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGTGAACGC
5301 TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGCGAAGC
5351 AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGAAT
5401 TAATTCCCCA GGCATCAAAT AAAACGAAAG GCTCAGTCGA AAGACTGGGC
5451 CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA CGCTCTCCTG AGTAGGACAA
5501 ATCCGCCGGG AGCGGATTTG AACGTTGCGA AGCAACGGCC CGGAGGGTGG
5551 CGGGCAGGAC GCCCGCCATA AACTGCCAGG AATTGGGGAT CGGAATTAAT
5601 TCCCGGTTTA AACCGGGGAT CTCGATCCCG CGAAATTAAT ACGACTCACT
5651 ATAGGGGAAT TGTGAGCGGA TAACAATTCC CCTCTAGAAA TAATTTTGTT
5701 TAACTTTAAG AAGGAGATAT ACATATGAAA ATCGAAGAAG GTAAACTGAC
5751 AAATCCTGGT GTATCCGCTT GGCAGGTCAA CACAGCTTAT ACTGCGGGAC
5801 AATTGGTCAC ATATAACGGC AAGACGTATA AATGTTTGCA GCCCCACACC
5851 TCCTTGGCAG GATGGGAACC ATCCAACGTT CCTGCCTTGT GGCAGCTTCA
5901 AAACAACGGT AACAACGGTC TCGAACTGCG CGAGTCCGGA GCTATCTCTG
5951 GCGATAGTCT GATCAGCCTG GCTAGCACAG GAAAAAGAGT TTCTATTAAA
6001 GATTTGTTAG ATGAAAAAGA TTTTGAAATA TGGGCAATTA ATGAACAGAC
6051 GATGAAGCTA GAATCAGCTA AAGTTAGTCG TGTATTTTGT ACTGGCAAAA
6101 AGCTAGTTTA TATTCTAAAA ACTCGACTAG GTAGAACTAT CAAGGCAACA
6151 GCAAATCATA GATTTTTAAC TATTGATGGT TGGAAAAGAT TAGATGAGCT
6201 ATCTTTAAAA GAGCATATTG CTCTACCCCG TAAACTAGAA AGCTCCTCTT
6251 TACAATTGTC ACCAGAAATA GAAAAGTTGT CTCAGAGTGA TATTTACTGG
6301 GACTCCATCG TTTCTATTAC GGAGACTGGA GTCGAAGAGG TTTTTGATTT
6351 GACTGTGCCA GGACCACATA ACTTTGTCGC GAATGACATC ATTGTACACA
6401 ACGGAAGAGC CATGGGCGGC CGCGAATTCC TCGAGGGCTC TTCCTGCATC
6451 ACGGGAGATG CACTAGTTGC CCTACCCGAG GGCGAGTCGG TACGCATCGC
6501 CGACATCGTG CCGGGTGCGC GGCCCAACAG TGACAACGCC ATCGACCTGA
6551 AAGTCCTTGA CCGGCATGGC AATCCCGTGC TCGCCGACCG GCTGTTCCAC
6601 TCCGGCGAGC ATCCGGTGTA CACGGTGCGT ACGGTCGAAG GTCTGCGTGT
6651 GACGGGCACC GCGAACCACC CGTTGTTGTG TTTGGTCGAC GTCGCCGGGG
6701 TGCCGACCCT GCTGTGGAAG CTGATCGACG AAATCAAGCC GGGCGATTAC
6751 GCGGTGATTC AACGCAGCGC ATTCAGCGTC GACTGTGCAG GTTTTGCCCG
6801 CGGAAAACCC GAATTTGCGC CCACAACCTA CACAGTCGGC GTCCCTGGAC
6851 TGGTGCGTTT CTTGGAAGCA CACCACCGAG ACCCGGACGC CCAAGCTATC
6901 GCCGACGAGC TGACCGACGG GCGGTTCTAC TACGCGAAAG TCGCCAGTGT
6951 CACCGACGCC GGCGTGCAGC CGGTGTATAG CCTTCGTGTC GACACGGCAG
7001 ACCACGCGTT TATCACGAAC GGGTTCGTCA GCCACGCTAC TGGCCTCACC
7051 GGTCTGAACT CAGGCCTCAC GACAAATCCT GGTGTATCCG CTTGGCAGGT
7101 CAACACAGCT TATACTGCGG GACAATTGGT CACATATAAC GGCAAGACGT
7151 ATAAATGTTT GCAGCCCCAC ACCTCCTTGG CAGGATGGGA ACCATCCAAC
7201 GTTCCTGCCT TGTGGCAGCT TCAATGACTG CAGGAAGGGG ATCCGGCTGC
7251 TAACAAAGCC CGAAAGGAAG CTGAGTTGGC TGCTGCCACC GCTGAGCAAT
7301 AACTAGCATA ACCCCTTGGG GCCTCTAAAC GGGTCTTGAG GGGTTTTTTG
7351 CTGAAAGGAG GAACTATATC CGGAT
