LOCUS       pMCSG61.        6422 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with a 6xHis-TEV-MBP leader, for 
            high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG61.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6422)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), amplify with LIC-F and LIC-R
            and treat with T4 DNA polymerase plus dTTP.
FEATURES             Location/Qualifiers
     source          1..6422
                     /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(256..1353)
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             complement(1360..1380)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(1405..1422)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1423..1425)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1433..1455)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1469..1493)
                     /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(1494..1512)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1825..1902
                     /label=lacI promoter
     CDS             1903..2982
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2998..3019
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3794..3982
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4087..4229
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4415..5003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5177..6034)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6035..6138)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcaccttct tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaatg ttatttttca ccgacgacaa
      241 ggatgctgct gcgttggttt gagcagcggc aagagcagca tcgacagtct gacgaccgct
      301 ggcggcgttg atcaccgcag tacgcacggc ataccagaaa gcggacatct gcgggatgtt
      361 cggcatgatt tcacctttct gggcgttttc catggtggcg gcaatacgtg gatctttcgc
      421 caactcttcc tcgtaagact tcagcgctac ggcacccagc ggtttgtctt tattaaccgc
      481 ttccagacct tcatcagtca gcagatagtt ttcgaggaac tctttcgcca gctctttgtt
      541 cggactggcg gcgttaatac ctgcgctcag cacgccaacg aacggtttgg atggttgacc
      601 cttgaaggtc ggcagtaccg ttacaccata attcactgcg ctggtgtcga tgttggacca
      661 tgcccacggg ccgttgatgg tcatcgctgt ttcgccttta ttaaaggcag cttctgcgat
      721 ggagtaatcg gtgtctgcat tcatgtgttt gtttttaatc aggtcaacca ggaaggtcag
      781 acccgctttc gcgccagcgt tatccacgcc cacgtcttta atgtcgtact tgcctgctgc
      841 atacttgaac gcataacccc cgtcagcagc aatcagcggc caggtgaagt acggttcttg
      901 caggttgaac atcagcgcgc tcttaccttt cgctttcagt tctttatcca gcgccgggat
      961 ctcttcccag gtttttggcg ggttcggcag cagatctttg ttataaatca gcgataacgc
     1021 ttcaacagcg atcgggtaag caatcagctt gccgttgtaa cgtacggcat cccaggtaaa
     1081 cggatacagc ttgtcctgga acgctgctgc cggggtgatt tcagccaaca ggccagattg
     1141 agcgtagcca ccaaagcggt cgtgtgccca gaagataatg tcagggccat cgccagttgc
     1201 cgcaacctgt gggaatttct cttccagttt atccggatgc tcaacggtga ctttaattcc
     1261 ggtatctttc tcgaatttct taccgacttc agcgagaccg ttatagcctt tatcgccgtt
     1321 aatccagatt accagtttac cttcttcgat tttggcattg gattggaagt acaggttctc
     1381 ggtacccaga tctacaccag aagaatgatg atgatgatgg tgcatatgta tatctccttc
     1441 ttaaagttaa acaaaattat tctagagggg aattgttatc cgctcacaat tcccctatag
     1501 tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg
     1561 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     1621 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     1681 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     1741 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     1801 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     1861 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     1921 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     1981 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     2041 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     2101 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     2161 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     2221 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     2281 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     2341 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     2401 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     2461 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     2521 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     2581 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     2641 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     2701 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     2761 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     2821 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     2881 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     2941 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3001 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     3061 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     3121 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     3181 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     3241 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     3301 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     3361 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     3421 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     3481 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3541 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3601 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3661 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3721 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     3781 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     3841 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3901 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3961 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4021 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4081 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     4141 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     4201 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     4261 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4321 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4381 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4441 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4501 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4561 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4621 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4681 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4741 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4801 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4861 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4921 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4981 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5041 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5101 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5161 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5221 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5281 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5341 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5401 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5461 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     5521 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5581 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5641 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5701 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5761 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5821 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5881 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5941 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6001 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa attattgaag
     6061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     6181 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6241 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6301 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6361 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     6421 ca
//
