LOCUS       pMCSG12.        4133 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-S- loop-TEV leader and a 
            p15A origin, for high-throughput purification of recombinant 
            proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG12
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4133)
  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 4133)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4133)
  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..4133
                     /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             339..392
                     /label=S loop
                     /note="GroES chaperone mobile loop that interacts with
                     GroEL"
     CDS             399..419
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             491..535
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      587..634
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(857..1513)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(1514..1616)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(2142..2686)
                     /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(2877..2898)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2914..3993)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3994..4071)
                     /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 tagatctgga agttgaaacc aaatctgctg
      361 gtggtatcgt tctgaccggt tctgctgctg ctggtaccga gaacctgtac ttccaatcca
      421 atattggaag tggataacgg atccgaattc gagctccgtc gacaagcttg cggccgcact
      481 cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg actcgtctac
      541 tagcgcagct taattaacct aggctgctgc caccgctgag caataactag cataacccct
      601 tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat ttgagaagca
      661 cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc
      721 tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca
      781 ttcatccgct tattatcact tattcaggcg tagcaccagg cgtttaaggg caccaataac
      841 tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa
      901 gcattctgcc gacatggaag ccatcacaga cggcatgatg aacctgaatc gccagcggca
      961 tcagcacctt gtcgccttgc gtatagtatt tgcccatagt gaaaacgggg gcgaagaagt
     1021 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     1081 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     1141 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     1201 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     1261 atatcaccag ctcaccgtct ttcattgcca tacggaactc cggatgagca ttcatcaggc
     1321 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     1381 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     1441 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     1501 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     1561 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt
     1621 ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta
     1681 tttattctgc gaagtgatct tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg
     1741 atgctgccaa cttactgatt tagtgtatga tggtgttttt gaggtgctcc agtggcttct
     1801 gtttctatca gctgtccctc ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc
     1861 accgccggac atcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg
     1921 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa
     1981 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     2041 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     2101 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
     2161 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
     2221 taaagatacc aggcgtttcc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     2281 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     2341 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     2401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     2461 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     2521 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     2581 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     2641 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     2701 taatcagata aattatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     2761 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     2821 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     2881 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2941 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3001 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3061 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3121 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3181 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3241 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3301 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3361 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     3421 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     3481 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     3541 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     3601 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     3661 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     3721 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     3781 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     3841 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     3901 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     3961 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4021 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4081 gacgctctcc cttatgcgac tcctgcatta ggaaattaat acgactcact ata
//
