LOCUS       pMCSG23.        4971 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial coexpression vector with a 6xHis-MBP-TEV leader and a 
            CloDF13 origin, for high-throughput purification of recombinant 
            proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG23
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4971)
  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 4971)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4971)
  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..4971
                     /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..1442
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1464..1484
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1556..1600
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      1652..1699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1873..2661)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(2662..2753)
                     /label=AmpR promoter
     rep_origin      complement(2801..3539)
                     /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(3715..3736)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3752..4831)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4832..4909)
                     /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 taccaaaatc gaagaaggta
      361 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      421 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
      481 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
      541 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
      601 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
      661 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
      721 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
      781 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
      841 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
      901 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
      961 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1021 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1081 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1141 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1201 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1261 cttacgagga agagttggcg aaagatccac gtattgccgc caccatggaa aacgcccaga
     1321 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1381 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1441 ctgattacga tatcccaggt accgagaacc tgtacttcca atccaatatt ggaagtggat
     1501 aacggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagt ctggtaaaga
     1561 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     1621 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1681 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     1741 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     1801 ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc ttgaacgaat
     1861 tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt ggacaaattc
     1921 ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat aagcctgtct
     1981 agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc agtcggcagc
     2041 gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg acaacgtaag
     2101 cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg ttaaggtttc
     2161 atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct ccgccgctgg
     2221 acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca gatcaatgtc
     2281 gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt ctccaaattg
     2341 cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac
     2401 ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt
     2461 gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca gcaaatcaat
     2521 atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta cggccagcaa
     2581 cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag tcgatacttc
     2641 ggcgatcacc gcttccctca tactcttcct ttttcattat tattgaagca tttatcaggg
     2701 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaatagctag
     2761 ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga cacatacaaa
     2821 gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa cagcagggcc
     2881 gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat aaacagacgc
     2941 ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag tacgggcgaa
     3001 acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct tgcgctctcc
     3061 tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac gggaagtgtg
     3121 gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt cgctccaagc
     3181 tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc ggtaactgtt
     3241 cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc attggtaact
     3301 gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt gtgcgccaaa
     3361 gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc agttaccacg
     3421 gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag gtggtttttt
     3481 cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3541 tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     3601 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     3661 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3721 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3781 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3841 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3901 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3961 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4021 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4081 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4141 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4201 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4261 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4321 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4381 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4441 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4501 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4561 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4621 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4681 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4741 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4801 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4861 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4921 cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat a
//
