LOCUS       pMCSG9.        6405 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHis-MBP-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG9.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6405)
  AUTHORS   Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH, 
            Collart FR, Joachimiak A.
  TITLE     An expression vector tailored for large-scale, high-throughput 
            purification of recombinant proteins.
  JOURNAL   Protein Expr. Purif. 2006;47:446-54.
  PUBMED    16497515
REFERENCE   2  (bases 1 to 6405)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6405)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2006"; volume: "47"; pages: "446-54"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..6405
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(223..243)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(265..1362)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(1387..1404)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1405..1407)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1415..1437)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1452..1476)
                     /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)."
     promoter        complement(1477..1495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1808..1885
                     /label=lacI promoter
     CDS             1886..2965
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2981..3002
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3777..3965
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4070..4212
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4398..4986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5160..6017)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6018..6121)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaata ttggattgga agtacaggtt
      241 ctcggtacct gggatatcgt aatcagtctg cgcgtctttc agggcttcat cgacagtctg
      301 acgaccgctg gcggcgttga tcaccgcagt acgcacggca taccagaaag cggacatctg
      361 cgggatgttc ggcatgattt cacctttctg ggcgttttcc atggtggcgg caatacgtgg
      421 atctttcgcc aactcttcct cgtaagactt cagcgctacg gcacccagcg gtttgtcttt
      481 attaaccgct tccagacctt catcagtcag cagatagttt tcgaggaact cttttgccag
      541 ctctttgttc ggactggcgg cgttaatacc tgcgctcagc acgccaacga acggtttgga
      601 tggttgaccc ttgaaggtcg gcagtaccgt tacaccataa ttcactttgc tggtgtcgat
      661 gttggaccat gcccacgggc cgttgatggt catcgctgtt tcgcctttat taaaggcagc
      721 ttctgcgatg gagtaatcgg tgtctgcatt catgtgtttg tttttaatca ggtcaaccag
      781 gaaggtcaga cccgctttcg cgccagcgtt atccacgccc acgtctttaa tgtcgtactt
      841 gccgttttca tacttgaacg cataaccccc gtcagcagca atcagcggcc aggtgaagta
      901 cggttcttgc aggttgaaca tcagcgcgct cttacctttc gctttcagtt ctttatccag
      961 cgccgggatc tcttcccagg tttttggcgg gttcggcagc agatctttgt tataaatcag
     1021 cgataacgct tcaacagcga tcgggtaagc aatcagcttg ccgttgtaac gtacggcatc
     1081 ccaggtaaac ggatacagct tgtcctggaa cgctttgtcc ggggtgattt cagccaacag
     1141 gccagattga gcgtagccac caaagcggtc gtgtgcccag aagataatgt cagggccatc
     1201 gccagttgcc gcaacctgtg ggaatttctc ttccagttta tccggatgct caacggtgac
     1261 tttaattccg gtatctttct cgaatttctt accgacttca gcgagaccgt tatagccttt
     1321 atcgccgtta atccagatta ccagtttacc ttcttcgatt ttggtaccca gatctacacc
     1381 agaagaatga tgatgatgat ggtgcatatg tatatctcct tcttaaagtt aaacaaaatt
     1441 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1501 gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1561 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1621 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1681 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1741 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1801 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1861 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1921 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1981 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     2041 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     2101 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     2161 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     2221 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     2281 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2341 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2401 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2461 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2521 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2581 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2641 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     2701 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2761 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2821 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2881 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2941 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     3001 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     3061 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     3121 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     3181 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     3241 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3301 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3361 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3421 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3481 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3541 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3601 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3661 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3721 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3781 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     3841 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3901 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3961 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4021 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     4081 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     4141 tgtactgaga gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     4201 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4261 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4321 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4381 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4441 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4501 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4561 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4621 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4741 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4801 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4861 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4921 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4981 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5041 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5101 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5161 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5221 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5281 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5341 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5401 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5461 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5521 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5581 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5641 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5701 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5761 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5821 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5881 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5941 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6001 aaatgttgaa tactcatact cttccttttt caaattattg aagcatttat cagggttatt
     6061 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6121 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatgtgaacc atcaccctaa
     6181 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6241 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6301 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6361 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
