LOCUS       pMCSG75.        5876 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding tRNA genes for rare Arg and Ile codons, 
            plus a C-terminal TEV-AviTag(TM)-6xHis cassette, plus the BirA 
            biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG75
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5876)
  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 5876)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5876)
  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 SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5876
                     /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(244..1206)
                     /label=BirA
                     /note="E. coli biotin protein ligase"
     RBS             1215..1220
                     /note="ribosome binding site"
     CDS             complement(1258..1275)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1282..1326)
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             complement(1333..1353)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     RBS             complement(1365..1385)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1401..1425)
                     /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(1426..1444)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1719..1795)
                     /label=argU
     tRNA            complement(2065..2140)
                     /label=ileX
     promoter        2280..2357
                     /label=lacI promoter
     CDS             2358..3437
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3453..3474
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3869..4457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4631..5488)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5489..5592)
                     /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 acttccaatg ttatggtggt gcccagctta
      241 ttatttttct gcactacgca gggatatttc accgcccatc cagggtttta ttattccatc
      301 ctgctcaagt aataaagccc cctgtttgtc tattccgcgt gaaatgccaa atatctcttt
      361 atcaccaatg ataagtttca ctgggcgatt aataaaatta tccagctttt cccagcgcga
      421 cagataaggt gccaatcctt cttgttcgaa gagttccaac gcagcacgta attcacgtat
      481 tagcatggcc gccaacgtat tacgatcgag attgatcccc gcttcctgca gcgtgatcca
      541 cccctgatta acgacactct cttcaacacg gcgcattgcc atgttgatcc cggctccaat
      601 gactatttgc gccgcatcgc cagttttgcc agtcagctcc acaagaatgc ctgccagctt
      661 gcgatcctgc agatagaggt cattaggcca tttgacacga actttatctg ctcccagctt
      721 gcgtaatact tccgccatca cgataccgat aaccagactt aaaccaatcg ccgccgccgg
      781 gccttgttcc agacgccaga acatcgacaa atataagttt gcgccaaaag gcgaaaacca
      841 tttccgcccc cggcgaccac ggccagcctg ctggtattct gcaacacagg catcgcccga
      901 tttaagctct ccgatacgat caagaaggta ctgattcgtg gagtcaataa ctggcagcac
      961 ggctacacta ccgccatcca gctgacccaa tatctgttca gcattaagta actggatggg
     1021 ctcaggcagg ctgtatcctt tacccggaac ggtaaagaca tcaacgcccc agtcacgcag
     1081 tgtctgaatg tgtttattaa tagccgcccg gctcattccc agcgtttcac ccaactgctc
     1141 gccagagtga aattcaccgt tcgctaacag ggcaatcaat ttcagtggca cggtgttatc
     1201 cttcatggga gagactcctt cttaaagtta agttatccac ttccaatgga tccgttaatg
     1261 atgatgatgg tggtgcccgg cttcatgcca ctcaattttt tgcgcctcaa aaatatcgtt
     1321 cagtccagag gcggattgga agtacaggtt ctccccggga gagactcctt cttaaagtta
     1381 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     1441 attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc
     1501 atcaccggtg gattcagtca ggcgtcccat tatcagtgct tcaggaaatg ggcggatggg
     1561 agtccataga aatggttcgt aggtatgctc accttgcgcc taatcatttg acagagcatg
     1621 cgaggaaaat agacgacatt tttggtgata atgtcccaaa tatgtcccac tctggaatta
     1681 tggaggatat aaagaaggcg taactgattg aattgtaatg gcgcgccctg caggattcga
     1741 acctgcggcc cacgacttag aaggtcgttg ctctatccaa ctgagctaag ggcgcgttga
     1801 taccgcaatg cggtgtaatc gcgtgaatta tacggtcaac ccttgctgag tcaatggctt
     1861 ttgatctcgc cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1921 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     1981 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcaaaa aggctgacga
     2041 tttctcgtca gcctttgcta tatctggtgg cccctgctgg acttgaacca gcgaccaagc
     2101 gattatgagt cgcctgctct aaccactgag ctaaggggcc atggtagcgg attataaagt
     2161 aactccgtgt cgcaatccgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     2221 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg
     2281 acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     2341 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     2401 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2461 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2521 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2581 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2641 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2701 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     2761 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     2821 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     2881 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     2941 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     3001 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     3061 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     3121 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     3181 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     3241 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     3301 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     3361 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3421 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     3481 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3541 cggggcatga ctacagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     3601 tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg
     3661 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4921 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaaattatt gaagcattta
     5521 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5581 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatgtgaac
     5641 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5701 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     5761 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     5821 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
