LOCUS       pMCSG51.        5346 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector for expressing a protein with a 
            6xHis-AviTag(TM)-TEV leader plus the BirA biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG51
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5346)
  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 5346)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5346)
  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..5346
                     /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(244..1206)
                     /label=BirA
                     /note="E. coli biotin protein ligase"
     RBS             1215..1220
                     /note="ribosome binding site"
     CDS             complement(1251..1271)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(1278..1322)
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             complement(1329..1346)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1347..1349)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1357..1379)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1394..1418)
                     /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(1419..1437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1750..1827
                     /label=lacI promoter
     CDS             1828..2907
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2923..2944
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3339..3927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4101..4958)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4959..5062)
                     /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 gcccagctta
      241 ttatttttct gcactacgca gggatatttc accgcccatc cagggtttta ttattccatc
      301 ctgctcaagt aataaagccc cctgtttgtc tattccgcgt gaaatgccaa atatctcttt
      361 atcaccaatg ataagtttca ctgggcgatt aataaaatta tccagctttt cccagcgcga
      421 cagataaggt gccaatcctt cttgttcgaa gagttccaac gcagcacgta attcacgtat
      481 tagcatggcc gccaacgtat tacgatcgag attgatcccc gcttcctgca gcgtgatcca
      541 cccctgatta acgacactct cttcaacacg gcgcattgcc atgttgatcc cggctccaat
      601 gactatttgc gccgcatcgc cagttttgcc agtcagctcc acaagaatgc ctgccagctt
      661 gcgatcctgc agatagaggt cattaggcca tttgacacga actttatctg ctcccagctt
      721 gcgtaatact tccgccatca cgataccgat aaccagactt aaaccaatcg ccgccgccgg
      781 gccttgttcc agacgccaga acatcgacaa atataagttt gcgccaaaag gcgaaaacca
      841 tttccgcccc cggcgaccac ggccagcctg ctggtattct gcaacacagg catcgcccga
      901 tttaagctct ccgatacgat caagaaggta ctgattcgtg gagtcaataa ctggcagcac
      961 ggctacacta ccgccatcca gctgacccaa tatctgttca gcattaagta actggatggg
     1021 ctcaggcagg ctgtatcctt tacccggaac ggtaaagaca tcaacgcccc agtcacgcag
     1081 tgtctgaatg tgtttattaa tagccgcccg gctcattccc agcgtttcac ccaactgctc
     1141 gccagagtga aattcaccgt tcgctaacag ggcaatcaat ttcagtggca cggtgttatc
     1201 cttcatggga gagactcctt cttaaagtta agttatccac ttccaatatt ggattggaag
     1261 tacaggttct cggtaccttc atgccactca attttttgcg cctcaaaaat atcgttcagt
     1321 ccagaagaat gatgatgatg atggtgcata tgtatatctc cttcttaaag ttaaacaaaa
     1381 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
     1441 tcgcgggatc gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     1501 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     1561 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     1621 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     1681 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     1741 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     1801 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     1861 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     1921 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     1981 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     2041 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     2101 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     2161 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     2221 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     2281 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     2341 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     2401 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     2461 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     2521 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     2581 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     2641 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     2701 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     2761 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     2821 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2881 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     2941 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     3001 ccggtgggcg cggggcatga ctacagtcac gtagcgatag cggagtgtat actggcttaa
     3061 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg
     3121 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3181 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3241 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3301 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3361 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3421 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3481 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3541 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3601 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3661 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3721 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3781 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3841 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3901 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3961 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4021 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4081 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4141 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4201 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4261 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4321 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4381 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     4441 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4501 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4561 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4621 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4681 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4741 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4801 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4861 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4921 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaaattatt
     4981 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5101 taatgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5161 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5221 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5281 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     5341 tcgcca
//
