LOCUS       pMCSG77.        5445 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a p15A origin encoding tRNA genes for rare Arg
            and Ile codons, for expressing a protein with a 6xHis-TEV leader 
            plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG77.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5445)
  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 5445)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5445)
  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     There are two sites for ligation-independent cloning (LIC).
            To express a 6xHis-TEV-tagged protein, linearize with SspI and treat
            with T4 DNA polymerase plus dGTP.
            To express a second untagged protein, linearize with SmaI and treat 
            with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5445
                     /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"
     RBS             243..248
                     /note="ribosome binding site"
     CDS             complement(279..299)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(324..341)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(342..344)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(352..374)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(389..413)
                     /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(414..432)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(707..783)
                     /label=argU
     tRNA            complement(1053..1128)
                     /label=ileX
     promoter        1245..1322
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1323..2402
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2418..2439
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     terminator      2607..2641
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(3353..4165)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(4760..5305)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
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 gcccgggaga
      241 gactccttct taaagttaag ttatccactt ccaatattgg attggaagta caggttctcg
      301 gtacccagat ctacaccaga agaatgatga tgatgatggt gcatatgtat atctccttct
      361 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
      421 tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg
      481 tggccggcat caccggtgga ttcagtcagg cgtcccatta tcagtgcttc aggaaatggg
      541 cggatgggag tccatagaaa tggttcgtag gtatgctcac cttgcgccta atcatttgac
      601 agagcatgcg aggaaaatag acgacatttt tggtgataat gtcccaaata tgtcccactc
      661 tggaattatg gaggatataa agaaggcgta actgattgaa ttgtaatggc gcgccctgca
      721 ggattcgaac ctgcggccca cgacttagaa ggtcgttgct ctatccaact gagctaaggg
      781 cgcgttgata ccgcaatgcg gtgtaatcgc gtgaattata cggtcaaccc ttgctgagtc
      841 aatggctttt gatctcgccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
      901 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
      961 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaaaaag
     1021 gctgacgatt tctcgtcagc ctttgctata tctggtggcc cctgctggac ttgaaccagc
     1081 gaccaagcga ttatgagtcg cctgctctaa ccactgagct aaggggccat ggtagcggat
     1141 tataaagtaa ctccgtgtcg caatccgcat gcgtcgactg tgtactcaac caagtcattc
     1201 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcgacacc atcgaatggt
     1261 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     1321 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     1381 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     1441 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     1501 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     1561 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     1621 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     1681 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     1741 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     1801 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     1861 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     1921 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     1981 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     2041 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     2101 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     2161 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     2221 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     2281 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     2341 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2401 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac
     2461 tttatgcttc cgggttttct ttgcctcacg atcgccccca aaacacataa ccaattgtat
     2521 ttattgaaaa ataaatagat acaactcact aaacatagca attcagatcc ggtccagtaa
     2581 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     2641 ttggtgagaa tcgcagcaac ttgtcgcgcc aatcgagcca tgtcgtcgtc aacgaccccc
     2701 cattcaagaa cagcaagcag cattgagaac tttggaatcc agtccctctt ccacctgctg
     2761 accggatcct ccggcgttca gcctgtgcca cagccgacag gatggtgacc accatttgcc
     2821 ccatatcacc gtcggtactg atcccgtcgt caataaaccg aaccgctaca ccctgagcat
     2881 caaactcttt tatcagttgg atcatgtcgg cggtgtcgcg gccaagacgg tcgagcttct
     2941 tcaccagaat gacatcacct tcctccacct tcatcctcag caaatccagc ccttcccgat
     3001 ctgttgaact gccggatgcc ttgtcggtaa agatgcggtt agcttttacc cctgcatctt
     3061 tgagcgctga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg cctgaatcgc
     3121 cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt tgtaggtgga
     3181 ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt cgggaagatg
     3241 cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt
     3301 caagtcagcg taatgctctg ccagtgttac aacaattaac caattctgat tagaaaaact
     3361 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3421 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3481 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3541 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3601 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3661 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3721 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3781 gcaggaacac tgccagcgca tcaacaatgt tttcacctga atcaggatat tcttctaata
     3841 cctggaatgc tgttttccgg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3901 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3961 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     4021 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     4081 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag
     4141 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4201 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     4261 acacaacgtg gctttgttga ataaatcgaa cttttgctga gttgaaggat cagatcacgc
     4321 atcttcccga caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc accaactggt
     4381 ccacctacaa caaagctctc atcaaccgtg gctccctcac tttctggctg gatgatgggg
     4441 cgattcaggc ctggtatgag tcagcaacac cttcttcacg aggcagacct cagcgctagc
     4501 ggagtgtata ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt
     4561 ggcaggagaa aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc
     4621 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct
     4681 tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga
     4741 gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct
     4801 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4861 ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc
     4921 tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc
     4981 tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt
     5041 aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact
     5101 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag
     5161 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc
     5221 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat
     5281 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aaggggtctg acgctcagtg
     5341 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5401 gatccttttt caaataggca agcatcacaa atgggtgttt gtaca
//
