LOCUS       pMCSG52.        5809 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding tRNA genes for rare Arg and Ile codons, 
            with a 6xHis-TEV leader for high-throughput purification of 
            recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG52.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5809)
  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 5809)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5809)
  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..5809
                     /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(268..285)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(286..288)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(296..318)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(333..357)
                     /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(358..376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(651..727)
                     /label=argU
     tRNA            complement(997..1072)
                     /label=ileX
     promoter        1212..1289
                     /label=lacI promoter
     CDS             1290..2369
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2385..2406
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3181..3369
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3474..3616
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3802..4390)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4564..5421)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5422..5525)
                     /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 ctcggtaccc agatctacac cagaagaatg atgatgatga tggtgcatat gtatatctcc
      301 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      361 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
      421 atcgtggccg gcatcaccgg tggattcagt caggcgtccc attatcagtg cttcaggaaa
      481 tgggcggatg ggagtccata gaaatggttc gtaggtatgc tcaccttgcg cctaatcatt
      541 tgacagagca tgcgaggaaa atagacgaca tttttggtga taatgtccca aatatgtccc
      601 actctggaat tatggaggat ataaagaagg cgtaactgat tgaattgtaa tggcgcgccc
      661 tgcaggattc gaacctgcgg cccacgactt agaaggtcgt tgctctatcc aactgagcta
      721 agggcgcgtt gataccgcaa tgcggtgtaa tcgcgtgaat tatacggtca acccttgctg
      781 agtcaatggc ttttgatctc gccggcgcca caggtgcggt tgctggcgcc tatatcgccg
      841 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
      901 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcaa
      961 aaaggctgac gatttctcgt cagcctttgc tatatctggt ggcccctgct ggacttgaac
     1021 cagcgaccaa gcgattatga gtcgcctgct ctaaccactg agctaagggg ccatggtagc
     1081 ggattataaa gtaactccgt gtcgcaatcc gcatgcacca ttccttgcgg cggcggtgct
     1141 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1201 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1261 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1321 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1381 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1441 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1501 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1561 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1621 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1681 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1741 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1801 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1861 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1921 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1981 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     2041 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     2101 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2161 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2221 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2281 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2341 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2401 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2461 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2521 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     2581 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     2641 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     2701 ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     2761 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     2821 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     2881 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     2941 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     3001 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     3061 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     3121 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     3181 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     3241 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     3301 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
     3361 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3421 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3481 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     3541 agattgtact gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     3601 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3661 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3721 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3781 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3841 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3901 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3961 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4021 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4081 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4141 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4201 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4261 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4321 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4381 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4441 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4501 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4561 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4621 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4681 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4741 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4801 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4861 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4921 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4981 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5041 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5101 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5161 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5221 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5281 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5341 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5401 acggaaatgt tgaatactca tactcttcct ttttcaaatt attgaagcat ttatcagggt
     5461 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5521 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatgtg aaccatcacc
     5581 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5641 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5701 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5761 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
