LOCUS       pMCSG22.        3906 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-S- loop-TEV leader and a 
            CloDF13 origin, for high-throughput purification of recombinant 
            proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG22
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3906)
  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 3906)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3906)
  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..3906
                     /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(808..1596)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(1597..1688)
                     /label=AmpR promoter
     rep_origin      complement(1736..2474)
                     /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(2650..2671)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2687..3766)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3767..3844)
                     /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 cgggtcatcg tggccggatc ttgcggcccc
      781 tcggcttgaa cgaattgtta gacattattt gccgactacc ttggtgatct cgcctttcac
      841 gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc
      901 aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca ggcgctccat
      961 tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat
     1021 gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca
     1081 tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag
     1141 ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat
     1201 agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg
     1261 ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg
     1321 cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt
     1381 ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt
     1441 aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa
     1501 atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag
     1561 ttgagtcgat acttcggcga tcaccgcttc cctcatactc ttcctttttc attattattg
     1621 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1681 taaacaaata gctagctcac tcggtcgcta cgctccgggc gtgagactgc ggcgggcgct
     1741 gcggacacat acaaagttac ccacagattc cgtggataag caggggacta acatgtgagg
     1801 caaaacagca gggccgcgcc ggtggcgttt ttccataggc tccgccctcc tgccagagtt
     1861 cacataaaca gacgcttttc cggtgcatct gtgggagccg tgaggctcaa ccatgaatct
     1921 gacagtacgg gcgaaacccg acaggactta aagatcccca ccgtttccgg cgggtcgctc
     1981 cctcttgcgc tctcctgttc cgaccctgcc gtttaccgga tacctgttcc gcctttctcc
     2041 cttacgggaa gtgtggcgct ttctcatagc tcacacactg gtatctcggc tcggtgtagg
     2101 tcgttcgctc caagctgggc tgtaagcaag aactccccgt tcagcccgac tgctgcgcct
     2161 tatccggtaa ctgttcactt gagtccaacc cggaaaagca cggtaaaacg ccactggcag
     2221 cagccattgg taactgggag ttcgcagagg atttgtttag ctaaacacgc ggttgctctt
     2281 gaagtgtgcg ccaaagtccg gctacactgg aaggacagat ttggttgctg tgctctgcga
     2341 aagccagtta ccacggttaa gcagttcccc aactgactta accttcgatc aaaccacctc
     2401 cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa ggatctcaag
     2461 aagatccttt gatcttttct actgaaccgc tctagatttc agtgcaattt atctcttcaa
     2521 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc
     2581 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     2641 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     2701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2761 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     2821 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     2881 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     2941 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     3001 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     3061 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     3121 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     3181 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     3241 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     3301 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     3361 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     3421 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     3481 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     3541 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     3601 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     3661 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     3721 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     3781 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     3841 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc
     3901 actata
//
