LOCUS       pMCSG63.        4864 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector 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    pMCSG63.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4864)
  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 4864)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4864)
  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..4864
                     /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        1268..1345
                     /label=lacI promoter
     CDS             1346..2425
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2441..2462
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2857..3445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3619..4476)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4477..4580)
                     /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 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 gcaccattcc ttgcggcggc ggtgctcaac
     1201 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1261 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1321 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1381 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1441 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1501 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1561 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1621 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     1681 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     1741 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     1801 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     1861 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     1921 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     1981 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2041 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2101 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     2161 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2221 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2281 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2341 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2401 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     2461 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2521 ggtgggcgcg gggcatgact acagtcacgt agcgatagcg gagtgtatac tggcttaact
     2581 atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg aaataccgca
     2641 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     2701 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2761 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     3301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3901 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     3961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4201 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aaattattga
     4501 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4561 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     4621 atgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4681 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4741 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4801 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     4861 gcca
//
