LOCUS       pMCSG24.        4527 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-GST-TEV leader and a 
            CloDF13 origin, for high-throughput purification of recombinant 
            proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG24
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4527)
  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 4527)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4527)
  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..4527
                     /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..992
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
     CDS             1020..1040
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1112..1156
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      1208..1255
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1429..2217)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(2218..2309)
                     /label=AmpR promoter
     rep_origin      complement(2357..3095)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     protein_bind    complement(3271..3292)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3308..4387)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4388..4465)
                     /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 tagatctgat gtcccctata ctaggttatt
      361 ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat
      421 atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat
      481 tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt
      541 ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag
      601 agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga
      661 gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg
      721 aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg
      781 taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa
      841 tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac
      901 aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag
      961 ccacgtttgg tggtggcgac catcctccaa aatcggatga ttacccagat ctgggtaccg
     1021 agaacctgta cttccaatcc aatattggaa gtggataacg gatccgaatt cgagctccgt
     1081 cgacaagctt gcggccgcac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg
     1141 ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga
     1201 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1261 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     1321 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcatc
     1381 gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt tgccgactac
     1441 cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc
     1501 caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata
     1561 ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc
     1621 ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc
     1681 ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg
     1741 ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc
     1801 tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc
     1861 tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg
     1921 ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct
     1981 ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc
     2041 atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc
     2101 atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat
     2161 gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact
     2221 cttccttttt cattattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2281 atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct acgctccggg
     2341 cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt ccgtggataa
     2401 gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt tttccatagg
     2461 ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc tgtgggagcc
     2521 gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt aaagatcccc
     2581 accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg
     2641 atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag ctcacacact
     2701 ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg
     2761 ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac ccggaaaagc
     2821 acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag gatttgttta
     2881 gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga
     2941 tttggttgct gtgctctgcg aaagccagtt accacggtta agcagttccc caactgactt
     3001 aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa aagattacgc
     3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg ctctagattt
     3121 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
     3181 aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     3241 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     3301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3421 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3481 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     3541 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     3601 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3661 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     3721 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3781 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3841 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     3901 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     3961 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4021 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4081 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4141 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4201 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4261 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     4321 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4381 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4441 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     4501 attaggaaat taatacgact cactata
//
