LOCUS       pMCSG53.        4808 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact 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    pMCSG53.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4808)
  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 4808)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4808)
  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..4808
                     /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."
     rep_origin      complement(2801..3389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3563..4420)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4421..4524)
                     /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 gactacagtc acgtagcgat agcggagtgt atactggctt
     2521 aactatgcgg catcagagca gattgtactg agagtgcacc atatatgcgg tgtgaaatac
     2581 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     2641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3241 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3541 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3601 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3661 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3721 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3781 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3841 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     3901 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3961 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4021 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4081 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4141 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4201 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4261 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4321 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4381 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaaatta
     4441 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4501 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     4561 gttaatgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     4621 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4681 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     4741 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     4801 attcgcca
//
