LOCUS       pMCSG64.        5366 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 6xHis-FLAG(R)-TEV 
            leader plus a second untagged protein.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG64.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5366)
  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 5366)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5366)
  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-FLAG(R)-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..5366
                     /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(306..329)
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             complement(348..365)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(366..368)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(376..398)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(413..437)
                     /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(438..456)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        769..846
                     /label=lacI promoter
     CDS             847..1926
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1942..1963
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2738..2926
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3031..3173
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3359..3947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4121..4978)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4979..5082)
                     /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 gtacccttat cgtcgtcatc cttataatcc agatctacac cagaagaatg atgatgatga
      361 tggtgcatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
      421 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct
      481 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
      541 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
      601 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
      661 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
      721 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
      781 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
      841 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
      901 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
      961 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     1021 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     1081 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     1141 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     1201 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     1261 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     1321 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     1381 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     1441 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     1501 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     1561 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     1621 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     1681 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     1741 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     1801 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     1861 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     1921 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     1981 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     2041 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     2101 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     2161 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     2221 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
     2281 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     2341 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     2401 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     2461 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     2521 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     2581 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     2641 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     2701 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     2761 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     2821 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     2881 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2941 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3001 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3061 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     3121 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     3181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaaattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5041 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5101 gccacctaaa ttgtaagcgt taatgtgaac catcacccta atcaagtttt ttggggtcga
     5161 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5221 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5281 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5341 cgctacaggg cgcgtcccat tcgcca
//
