LOCUS       pTWIN2.        7192 bp DNA     circular SYN 09-DEC-2020
DEFINITION  Cloning vector pTWIN2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    pTWIN2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7192)
  AUTHORS   Evans TC Jr., Benner J, 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 TC Jr., Chute IC, Wu H, Chong S, Benner J, Liu X-Q.,
            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 TC Jr., Benner J, 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 FB.
  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, Xu MQ.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-OCT-2007) Research Department, New England Biolabs, 
            240 County Road, Ipswich, MA 01938, USA
REFERENCE   6  (bases 1 to 7192)
  TITLE     Direct Submission
REFERENCE   7  (bases 1 to 7192)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol Chem
            274, 3923-3926 (1999)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Gene 231, 
            1-13 (1999)"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "J Biol Chem
            274, 18359-18363 (1999)"
COMMENT     SGRef: number: 4; type: "Journal Article"; journalName: "Nucleic 
            Acids Res"; date: "2002"; volume: "30"; issue: "1"; pages: "383-384"
COMMENT     SGRef: number: 5; type: "Journal Article"; journalName: "Submitted 
            (23-OCT-2007) Research Department, New England Biolabs, 240 County 
            Road, Ipswich, MA 01938, USA"
COMMENT     SGRef: number: 6; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..139
                     /label=AmpR promoter
     CDS             140..997
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      complement(1042..1555)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     rep_origin      1666..2254
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2626..2814)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3337..3358)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3374..4453)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4454..4531)
                     /label=lacI promoter
     terminator      4684..4770
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(5419..5462)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        5637..5655
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5656..5680
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5695..5717
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5749..5901
                     /label=CBD
                     /note="chitin binding domain from chitinase A1 (Watanabe et
                     al., 1994)"
     primer_bind     6296..6315
                     /note="Ssp DnaB intein forward primer sequence (NEB
                     #S1269S)"
     misc_feature    6403..6444
                     /gene="expression CDS"
                     /label=multiple cloning site (SapI-SapI)
                     /note="multiple cloning site (SapI-SapI)"
     primer_bind     complement(6505..6522)
                     /note="Mth RIR1 intein reverse primer sequence (NEB
                     #S1270S)"
     terminator      7120..7167
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
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
//
