LOCUS       pMCSG13.        5198 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-MBP-TEV leader and a p15A
            origin, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG13
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5198)
  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 5198)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5198)
  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..5198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             71..73
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             83..100
                     /label=6xHis
                     /note="6xHis affinity tag"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /label=lac repressor encoded by lacI binding site
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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             286..291
                     /note="ribosome binding site"
     CDS             300..302
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             303..320
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             345..1442
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1464..1484
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1556..1600
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      1652..1699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1922..2578)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(2579..2681)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(3207..3751)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     protein_bind    complement(3942..3963)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3979..5058)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(5059..5136)
                     /label=lacI promoter
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgcaccatca tcatcatcat tcttctggtg tagatctggg taccaaaatc gaagaaggta
      361 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      421 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
      481 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
      541 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
      601 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
      661 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
      721 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
      781 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
      841 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
      901 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
      961 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1021 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1081 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1141 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1201 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1261 cttacgagga agagttggcg aaagatccac gtattgccgc caccatggaa aacgcccaga
     1321 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1381 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1441 ctgattacga tatcccaggt accgagaacc tgtacttcca atccaatatt ggaagtggat
     1501 aacggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagt ctggtaaaga
     1561 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     1621 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1681 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     1741 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     1801 ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat ccgcttatta
     1861 tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct taaaaaaatt
     1921 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     1981 ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     2041 cttgcgtata gtatttgccc atagtgaaaa cgggggcgaa gaagttgtcc atattggcca
     2101 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     2161 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     2221 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     2281 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     2341 cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca agaatgtgaa
     2401 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     2461 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     2521 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt
     2581 tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta
     2641 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa
     2701 agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt
     2761 gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac
     2821 tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt
     2881 ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag
     2941 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc
     3001 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat
     3061 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     3121 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     3181 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     3241 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     3301 tttcccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
     3361 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
     3421 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
     3481 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
     3541 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
     3601 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
     3661 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
     3721 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaatta
     3781 tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     3841 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     3901 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     3961 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4021 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4081 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4141 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4201 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     4261 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4321 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4381 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4441 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4501 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4561 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4621 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4681 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4741 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4801 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4861 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4921 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4981 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5041 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5101 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5161 gcgactcctg cattaggaaa ttaatacgac tcactata
//
