LOCUS       pMCSG56.        4332 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector with a 6xHis-2xHA-TEV leader for 
            high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG56.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4332)
  AUTHORS   Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, 
            Jedrzejczak R, Joachimiak A.
  TITLE     New LIC vectors for production of proteins from genes containing 
            rare codons.
  JOURNAL   J. Struct. Funct. Genomics 2013;14:135-44.
  PUBMED    24057978
REFERENCE   2  (bases 1 to 4332)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4332)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
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..4332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(223..243)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(250..276)
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(277..303)
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(322..339)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(340..342)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(350..372)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(387..411)
                     /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)."
     promoter        complement(412..430)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        743..820
                     /label=lacI promoter
     CDS             821..1900
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1916..1937
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2325..2913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3087..3944)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3945..4048)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaata ttggattgga agtacaggtt
      241 ctcggtacct gcgtagtccg gtacgtcgta cggatatgcg tagtccggca catcatacgg
      301 atacagatct acaccagaag aatgatgatg atgatggtgc atatgtatat ctccttctta
      361 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
      421 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
      481 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
      541 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
      601 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
      661 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
      721 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
      781 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
      841 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
      901 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
      961 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1021 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     1081 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     1141 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     1201 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     1261 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     1321 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     1381 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     1441 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     1501 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     1561 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     1621 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     1681 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     1741 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     1801 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1861 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     1921 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     1981 cagtcagctc cttccggtgg gcgcggggca tgactagtag cgatagcgga gtgtatactg
     2041 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa
     2101 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     2161 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2221 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2281 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2341 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2401 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2461 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2521 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2581 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2641 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2701 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2761 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2821 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2881 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2941 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3001 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3061 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3121 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3181 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3241 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3301 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3361 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     3421 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3481 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3541 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3601 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3661 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3721 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3781 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3841 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3901 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3961 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4021 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     4081 aagcgttaat gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     4141 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4201 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4261 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4321 tcccattcgc ca
//
