LOCUS       pMCSG21.        3852 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-TEV leader and a CloDF13 
            origin, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG21
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3852)
  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 3852)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3852)
  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..3852
                     /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..365
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             437..481
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      533..580
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(754..1542)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(1543..1634)
                     /label=AmpR promoter
     rep_origin      complement(1682..2420)
                     /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(2596..2617)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2633..3712)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3713..3790)
                     /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 taccgagaac ctgtacttcc
      361 aatccaatat tggaagtgga taacggatcc gaattcgagc tccgtcgaca agcttgcggc
      421 cgcactcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
      481 gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata
      541 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
      601 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
      661 agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc
      721 ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
      781 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
      841 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
      901 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
      961 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     1021 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     1081 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     1141 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     1201 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     1261 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     1321 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     1381 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     1441 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     1501 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcatta
     1561 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1621 gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
     1681 ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat
     1741 gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc
     1801 agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat
     1861 gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg
     1921 tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
     1981 ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg
     2041 tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct
     2101 gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac
     2161 tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt
     2221 gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
     2281 ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac
     2341 cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat
     2401 ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct
     2461 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     2521 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     2581 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     2641 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2701 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     2761 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     2821 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     2881 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     2941 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     3001 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     3061 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3121 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3181 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3241 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     3301 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     3361 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     3421 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     3481 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     3541 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     3601 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     3661 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     3721 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     3781 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     3841 cgactcacta ta
//
