LOCUS       pMCSG50.        6347 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing a protein with a 
            6xHis-AviTag(TM)-TEV leader plus the BirA biotin ligase.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG50.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6347)
  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 6347)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6347)
  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..6347
                     /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."
     CDS             3719..3907
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4012..4154
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4340..4928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5102..5959)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5960..6063)
                     /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 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     3061 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     3121 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     3181 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     3241 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     3301 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     3361 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     3421 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     3481 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     3541 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     3601 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     3661 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     3721 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     3781 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     3841 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     3901 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3961 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     4021 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     4081 attgtactga gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4141 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4741 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5401 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5941 ggaaatgttg aatactcata ctcttccttt ttcaaattat tgaagcattt atcagggtta
     6001 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6061 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatgtgaa ccatcaccct
     6121 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6181 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     6241 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6301 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
