LOCUS       pMCSG10.        5961 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a 6xHis-GST-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG10
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5961)
  AUTHORS   Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID.
  TITLE     A family of LIC vectors for high-throughput cloning and purification
            of proteins.
  JOURNAL   Methods Mol. Biol. 2009;498:105-15.
  PUBMED    18988021
REFERENCE   2  (bases 1 to 5961)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5961)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods 
            Mol. Biol."; date: "2009"; volume: "498"; pages: "105-15"
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..5961
                     /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(271..924)
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
     CDS             complement(943..960)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(961..963)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="Feature 14"
                     /translation="M"
     RBS             complement(971..993)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1008..1032)
                     /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(1033..1051)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1364..1441
                     /label=lacI promoter
     CDS             1442..2521
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2537..2558
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3333..3521
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3626..3768
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3954..4542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4716..5573)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5574..5677)
                     /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 agatctgggt aatcatccga ttttggagga tggtcgccac caccaaacgt
      301 ggcttgccag ccctgcaaag gccatgctat atacttgctg gatttcaagt acttatcaat
      361 ttgtgggata gcttcaatac gttttttaaa acaaactaat tttgggaacg catccaggca
      421 cattgggtcc atgtataaaa caacatcaag agcgtcatac aacatgaagt caggatgggt
      481 tacatgatca ccatttaaat atgttttatg acataaacga tcttcgaaca ttttcagcat
      541 ttcaggtagc ttgctaagaa aatcaacttt gagagtttca aagtctttac tatatgcaat
      601 tctcgaaaca ccgtatctaa tatccaaaac cgctccttca agcattgaaa tctctgcacg
      661 ctcttttgga caaccaccca acatgttgtg cttgtcagct atataacgta tgatggccat
      721 agactgtgtt aatttaacat caccatcaat ataataagga agattgggaa actccaaacc
      781 caattcaaac tttttgtttc gccatttatc accttcatcg cgctcataca aatgctcttc
      841 atatttttct tcaagatatt ccaaaagaag tcgagtgggt tgcacaaggc ccttaatttt
      901 ccaataacct agtatagggg acatcagatc tacaccagaa gaatgatgat gatgatggtg
      961 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1021 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     1081 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1141 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1201 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     1261 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     1321 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     1381 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1441 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1501 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1561 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1621 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1681 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1741 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1801 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1861 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1921 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     1981 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2041 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2101 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2161 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2221 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     2281 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2341 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2401 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2461 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2521 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     2581 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     2641 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     2701 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     2761 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     2821 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     2881 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     2941 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     3001 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     3061 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3121 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3181 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     3241 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     3301 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     3361 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     3421 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3481 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3541 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3601 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3661 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg
     3721 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     3781 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3841 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3901 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3961 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4021 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4081 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4141 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4201 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4261 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4321 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4381 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4441 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4501 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4561 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4621 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4681 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4741 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4801 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4861 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4921 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4981 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     5041 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5101 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5161 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5221 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5281 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5341 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5401 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5461 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5521 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5581 ctttttcaaa ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5641 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5701 ctaaattgta agcgttaatg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5761 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5821 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5881 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5941 cagggcgcgt cccattcgcc a
//
