LOCUS       pMCSG7.        5286 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHis-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG7.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5286)
  AUTHORS   Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
  TITLE     A new vector for high-throughput, ligation-independent cloning 
            encoding a tobacco etch virus protease cleavage site.
  JOURNAL   Protein Expr. Purif. 2002;25:8-15.
  PUBMED    12071693
REFERENCE   2  (bases 1 to 5286)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5286)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2002"; volume: "25"; pages: "8-15"
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..5286
                     /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(268..285)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(286..288)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(296..318)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(333..357)
                     /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(358..376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        689..766
                     /label=lacI promoter
     CDS             767..1846
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1862..1883
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2658..2846
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    2951..3093
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3279..3867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4041..4898)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4899..5002)
                     /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 ctcggtaccc agatctacac cagaagaatg atgatgatga tggtgcatat gtatatctcc
      301 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      361 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
      421 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
      481 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
      541 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
      601 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
      661 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
      721 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
      781 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
      841 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
      901 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
      961 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     1021 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     1081 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     1141 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     1201 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     1261 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     1321 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     1381 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     1441 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     1501 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     1561 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     1621 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     1681 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     1741 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     1801 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     1861 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     1921 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1981 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     2041 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     2101 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     2161 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     2221 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     2281 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     2341 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     2401 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     2461 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     2521 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     2581 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     2641 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     2701 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     2761 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     2821 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2881 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2941 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     3001 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg
     3061 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4321 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     4381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaaattatt
     4921 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4981 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5041 taatgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5101 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5161 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5221 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     5281 tcgcca
//
