LOCUS       pMCSG14.        4754 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-GST-TEV leader and a p15A
            origin, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG14
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4754)
  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 4754)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4754)
  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..4754
                     /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(1478..2134)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(2135..2237)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(2763..3307)
                     /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(3498..3519)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3535..4614)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4615..4692)
                     /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 ccgggtcgaa
     1381 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaccag
     1441 gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc
     1501 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat
     1561 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtatagtat ttgcccatag
     1621 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     1681 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     1741 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     1801 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     1861 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaact
     1921 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     1981 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     2041 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     2101 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     2161 tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac
     2221 ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat
     2281 caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt
     2341 cggcgcaaag tgcgtcgggt gatgctgcca acttactgat ttagtgtatg atggtgtttt
     2401 tgaggtgctc cagtggcttc tgtttctatc agctgtccct cctgttcagc tactgacggg
     2461 gtggtgcgta acggcaaaag caccgccgga catcagcgct agcggagtgt atactggctt
     2521 actatgttgg cactgatgag ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc
     2581 tgcaccggtg cgtcagcaga atatgtgata caggatatat tccgcttcct cgctcactga
     2641 ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga
     2701 tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg
     2761 tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg
     2821 tggcgaaacc cgacaggact ataaagatac caggcgtttc ccctggcggc tccctcgtgc
     2881 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     2941 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     3001 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3061 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
     3121 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
     3181 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
     3241 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
     3301 atctcaagaa gatcatctta ttaatcagat aaattatttc tagatttcag tgcaatttat
     3361 ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta
     3421 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3481 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     3541 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3601 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3661 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     3721 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     3781 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     3841 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     3901 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     3961 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4021 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4081 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4141 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4201 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4261 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4321 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4381 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     4441 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     4501 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     4561 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     4621 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     4681 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa
     4741 tacgactcac tata
//
