LOCUS       pMCSG60.        5342 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 6xHis-TEV leader 
            plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG60.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5342)
  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 5342)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5342)
  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..5342
                     /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"
     promoter        745..822
                     /label=lacI promoter
     CDS             823..1902
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1918..1939
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2714..2902
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3007..3149
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3335..3923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4097..4954)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4955..5058)
                     /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 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
      541 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
      601 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
      661 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
      721 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
      781 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
      841 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
      901 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
      961 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     1021 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     1081 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     1141 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     1201 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     1261 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     1321 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1381 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1441 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     1501 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     1561 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     1621 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     1681 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     1741 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     1801 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1861 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     1921 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     1981 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     2041 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     2101 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     2161 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     2221 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2281 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2341 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2401 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     2461 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     2521 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     2581 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     2641 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     2701 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     2761 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     2821 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     2881 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2941 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3001 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3061 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     3121 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3181 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3241 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3301 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3361 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3421 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3481 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3541 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3601 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3661 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3721 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3781 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3841 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3901 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3961 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4021 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4081 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4141 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4201 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4261 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4321 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4381 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     4441 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4501 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4561 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4621 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4681 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4741 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4801 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4861 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4921 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa attattgaag
     4981 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5041 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5101 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5161 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5221 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5281 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     5341 ca
//
