LOCUS       pMCSG76.        4310 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with a CloDF13 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    pMCSG76.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4310)
  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 4310)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4310)
  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..4310
                     /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
     CDS             complement(1233..2021)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(2022..2113)
                     /label=AmpR promoter
     rep_origin      complement(2161..2899)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     protein_bind    complement(3075..3096)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3112..4191)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4192..4269)
                     /label=lacI 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 gcgacccggt catcgtggcc ggatcttgcg
     1201 gcccctcggc ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct
     1261 ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct
     1321 tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc
     1381 tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac
     1441 caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag
     1501 ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca
     1561 aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc
     1621 aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg
     1681 cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg
     1741 tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc
     1801 gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc
     1861 accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg
     1921 tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct
     1981 gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcattat
     2041 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2101 aaaaataaac aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg
     2161 gcgctgcgga cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg
     2221 tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca
     2281 gagttcacat aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg
     2341 aatctgacag tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt
     2401 cgctccctct tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt
     2461 tctcccttac gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt
     2521 gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg
     2581 cgccttatcc ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact
     2641 ggcagcagcc attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg
     2701 ctcttgaagt gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc
     2761 tgcgaaagcc agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc
     2821 acctccccag gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc
     2881 tcaagaagat cctttgatct tttctactga accgctctag atttcagtgc aatttatctc
     2941 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc
     3001 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3061 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3121 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3181 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3241 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3301 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3361 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     3421 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3481 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3541 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3601 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3661 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3721 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     3781 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     3841 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     3901 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     3961 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4021 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4081 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4141 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4201 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4261 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
//
