LOCUS pTWIN2 7192 bp DNA circular 09-NOV-2011 DEFINITION Cloning vector pTWIN2, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pTWIN2 ORGANISM Cloning vector pTWIN2 other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 7192) AUTHORS Evans,T.C. Jr., Benner,J. and Xu,M.-Q. TITLE The in vitro ligation of bacterially expressed protein using an intein from Methanobacterium thermoautotrophicum JOURNAL J Biol Chem 274, 3923-3926 (1999) PUBMED 9933578 REFERENCE 2 (bases 1 to 7192) AUTHORS Mathys,S., Evans,T.C. Jr., Chute,I.C., Wu,H., Chong,S., Benner,J., Liu,X.-Q. and Xu,M.-Q. TITLE 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 JOURNAL Gene 231, 1-13 (1999) PUBMED 10231563 REFERENCE 3 (bases 1 to 7192) AUTHORS Evans,T.C. Jr., Benner,J. and Xu,M.-Q. TITLE The cyclization and polymerization of bacterially-expressed proteins using modified self-splicing inteins JOURNAL J Biol Chem 274, 18359-18363 (1999) PUBMED 10373440 REFERENCE 4 (bases 1 to 7192) AUTHORS Perler,F.B. TITLE InBase, the intein database JOURNAL Nucleic Acids Res 30 (1), 383-384 (2002) PUBMED 11752343 REFERENCE 5 (bases 1 to 7192) AUTHORS Ghosh,I. and Xu,M.Q. TITLE Direct Submission JOURNAL Submitted (23-OCT-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA FEATURES Location/Qualifiers source 1..7192 /organism="Cloning vector pTWIN2" /mol_type="other DNA" gene 140..1000 /gene="bla" CDS 140..1000 /gene="bla" /note="ampR (confers resistance to ampicillin)" /codon_start=1 /product="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" sig_peptide 140..208 /gene="bla" /note="required for secretion to the periplasm; cleaved off to form the mature beta-lactamase protein" rep_origin 1042..1555 /note="M13 origin of replication (+ -)" rep_origin 1666..2254 /note="pMB1 origin of replication (clockwise) (RNAII -35 to RNA/DNA switch point)" gene complement(2623..2814) /gene="rop" CDS complement(2623..2814) /gene="rop" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="ROP protein" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH ADELYRSCLARFGDDGENL" gene complement(3371..4453) /gene="lacI" CDS complement(3371..4453) /gene="lacI" /exception="alternative start codon" /codon_start=1 /transl_table=11 /product="lac repressor (LacI)" /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" primer_bind 5637..5656 /note="T7 universal primer sequence (NEB #S1248S)" promoter 5637..5654 /note="T7 promoter (transcript start 5654 clockwise)" protein_bind 5656..5680 /note="lac operator" /bound_moiety="LacI" RBS 5710..5716 /note="RBS for expression CDS" gene 5725..7044 /gene="expression CDS" CDS 5725..7044 /gene="expression CDS" /note="chitin binding domain (CBD) sequence 5725..5901; Ssp DNAB intein sequence 5941..6402; multiple cloning site (SapI-SapI) 6403..6444; Mth RIR1 intein sequence 6445..6846; CBD sequence 6886..7044" /codon_start=1 /product="CBD/intein/intein/CBD fusion" /translation="MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSL AGWEPSNVPALWQLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEI WAINEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELS LKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFV ANDIIVHNGRAMGGREFLEGSSCVSGDTIVMTSGGPRTVAELEGKPFTALIRGSGYPC PSGFFRTCERDVYDLRTREGHCLRLTHDHRVLVMDGGLEWRAAGELERGDRLVMDDAA GEFPALATFRGLRGAGRQDVYDATVYGASAFTANGFIVHACGEQPGTGLNSGLTTNPG VSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ" primer_bind 6296..6315 /note="Ssp DnaB intein forward primer sequence (NEB #S1269S)" misc_feature 6403..6444 /gene="expression CDS" /note="multiple cloning site (SapI-SapI)" primer_bind complement(6505..6522) /note="Mth RIR1 intein reverse primer sequence (NEB #S1270S)" terminator 7064..7186 /note="T7 Tphi transcription terminator" BASE COUNT 1785 a 1803 c 1934 g 1670 t 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 ttcctgcgta tccggtgaca ccattgtaat gactagtggc 6481 ggtccgcgca ctgtggctga actggagggc aaaccgttca ccgcactgat tcgcggctct 6541 ggctacccat gcccctcagg tttcttccgc acctgtgaac gtgacgtata tgatctgcgt 6601 acacgtgagg gtcattgctt acgtttgacc catgatcacc gtgttctggt gatggatggt 6661 ggcctggaat ggcgtgccgc gggtgaactg gaacgcggcg accgcctggt gatggatgat 6721 gcagctggcg agtttccggc actggcaacc ttccgtggcc tgcgtggcgc tggccgccag 6781 gatgtttatg acgctactgt ttacggtgct agcgcattca ctgctaatgg cttcattgta 6841 cacgcatgcg gcgagcagcc cgggaccggt ctgaactcag gcctcacgac aaatcctggt 6901 gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc 6961 aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt 7021 cctgccttgt ggcagcttca atgactgcag gaaggggatc cggctgctaa caaagcccga 7081 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 7141 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at //