LOCUS       pMCSG68.        4832 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding tRNA genes for rare Arg and Ile 
            codons, with a 6xHis-Strep-Tag(R) II-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG68
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4832)
  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 4832)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4832)
  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..4832
                     /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(250..273)
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             complement(292..309)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(310..312)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(320..342)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(357..381)
                     /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(382..400)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(675..751)
                     /label=argU
     tRNA            complement(1021..1096)
                     /label=ileX
     promoter        1236..1313
                     /label=lacI promoter
     CDS             1314..2393
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2409..2430
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2825..3413)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3587..4444)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4445..4548)
                     /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 ttttcgaact gcggatgaga ccacagatct acaccagaag aatgatgatg
      301 atgatggtgc atatgtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
      361 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatcg
      421 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggtggatt cagtcaggcg
      481 tcccattatc agtgcttcag gaaatgggcg gatgggagtc catagaaatg gttcgtaggt
      541 atgctcacct tgcgcctaat catttgacag agcatgcgag gaaaatagac gacatttttg
      601 gtgataatgt cccaaatatg tcccactctg gaattatgga ggatataaag aaggcgtaac
      661 tgattgaatt gtaatggcgc gccctgcagg attcgaacct gcggcccacg acttagaagg
      721 tcgttgctct atccaactga gctaagggcg cgttgatacc gcaatgcggt gtaatcgcgt
      781 gaattatacg gtcaaccctt gctgagtcaa tggcttttga tctcgccggc gccacaggtg
      841 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
      901 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
      961 tgggcgccat ctccttgcat gcaaaaaggc tgacgatttc tcgtcagcct ttgctatatc
     1021 tggtggcccc tgctggactt gaaccagcga ccaagcgatt atgagtcgcc tgctctaacc
     1081 actgagctaa ggggccatgg tagcggatta taaagtaact ccgtgtcgca atccgcatgc
     1141 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     1201 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     1261 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     1321 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     1381 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     1441 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     1501 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     1561 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     1621 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     1681 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     1741 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     1801 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     1861 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     1921 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     1981 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     2041 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     2101 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     2161 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     2221 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     2281 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     2341 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     2401 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     2461 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactac agtcacgtag
     2521 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     2581 caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     2641 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2701 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2761 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2821 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2881 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2941 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3001 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3061 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3121 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3181 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3241 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     3301 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3361 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3421 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3481 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3541 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3601 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3661 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3721 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3781 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3841 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     3901 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3961 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4021 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4081 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4141 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4201 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4261 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4321 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4381 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4441 tcatactctt cctttttcaa attattgaag catttatcag ggttattgtc tcatgagcgg
     4501 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4561 aaaagtgcca cctaaattgt aagcgttaat gtgaaccatc accctaatca agttttttgg
     4621 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4681 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4741 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4801 atgcgccgct acagggcgcg tcccattcgc ca
//
