LOCUS       pVP56A.        7222 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with an ampicillin resistance marker, for 
            appending an N-terminal 8xHis-MBP-HRV 3C-TEV cassette to an 
            expressed protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP56A.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7222)
  AUTHORS   Blommel PG, Martin PA, Wrobel RL, Steffen E, Fox BG.
  TITLE     High efficiency single step production of expression plasmids from 
            cDNA clones using the Flexi Vector cloning system.
  JOURNAL   Protein Expr. Purif. 2006;47:562-70.
  PUBMED    16377204
REFERENCE   2  (bases 1 to 7222)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7222)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2006"; volume: "47"; pages: "562-70"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     To remove the lethal barnase gene, insert an ORF between the SgfI 
            site and the PmeI site or a downstream site.
FEATURES             Location/Qualifiers
     source          1..7222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        46..90
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    98..114
                     /label=lac repressor encoded by lacI binding site
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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)."
     RBS             137..142
                     /note="ribosome binding site"
     CDS             151..153
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             157..180
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             187..1284
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1309..1332
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             1333..1353
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1382..1714
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        1733..1763
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1817..2473
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2486..2541
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2626..2646)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      2713..2807
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3577..3663
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3722..3743)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3759..4838)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4839..4916)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     misc_feature    5108..5248
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5434..6022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6196..7053)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7054..7158)
                     /label=AmpR promoter
ORIGIN
        1 aaaaataggc gtatcacgag gccctttcgt cttcacctcg agaaatcata aaaaatttat
       61 ttgctttgtg agcggataac aattataata gattcaattg tgagcggata acaatttcac
      121 acagaattca ttaaagagga gaaattaacc atgggacatc accatcatca ccatcaccat
      181 gcatccaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      241 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      301 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      361 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      421 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      481 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      541 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      601 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      661 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
      721 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      781 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      841 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
      901 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
      961 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     1021 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1081 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     1141 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1201 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1261 gaagccctga aagacgcgca gactttaatt aacggcgacg gtgccgggct ggaagttctg
     1321 ttccaggggc ccgaaaacct gtacttccag gcgatcgcca tggaataagt aaaggaatca
     1381 catggcacag gttatcaaca cgtttgacgg ggttgcggat tatcttcaga catatcataa
     1441 gctacctgat aattacatta caaaatcaga agcacaagcc ctcggctggg tggcatcaaa
     1501 agggaacctt gcagacgtcg ctccggggaa aagcatcggc ggagacatct tctcaaacag
     1561 ggaaggcaaa ctcccgggca aaagcggacg aacatggcgt gaagcggata ttaactatac
     1621 atcaggcttc agaaattcag accggattct ttactcaagc gactggctga tttacaaaac
     1681 aacggaccat tatcagacct ttacaaaaat cagataatta ggcaccccag gctttacact
     1741 ttatgctttc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg
     1801 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     1861 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     1921 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     1981 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
     2041 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     2101 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     2161 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     2221 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     2281 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     2341 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     2401 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     2461 gcagggcggg gcgtaatgtt taaacgaatt cgagctcggt acccggggat cctctagagt
     2521 cgacctgcag gcatgcaagc tgatccggct gctaacaaag cccgaaagga agctgagttg
     2581 gctgctgcca ccgctgagca ataactagca aactcgtttc tcgttcagct ttcttgtaca
     2641 aagtggtgat ggcgcgcctg taggacgtcg acggtaccat cgatacgcgt tcgaagctta
     2701 atagctgagc tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     2761 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg
     2821 gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     2881 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     2941 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     3001 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     3061 ccggaaattt cgtatggcaa tgaaagacgg tgagctggtg atatggggat agtgttcacc
     3121 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     3181 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     3241 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     3301 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     3361 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     3421 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     3481 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     3541 aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg
     3601 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     3661 aatccgccct ctagattacg tgcagtcgat gataagctgt caaacatgag aattgtgcct
     3721 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3901 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3961 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4021 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4081 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4141 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4201 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4261 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4321 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4381 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4441 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4501 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4561 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4621 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4681 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4741 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4801 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4861 tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcggaa
     4921 tttcgggcag cgttgggtcc tggccacggg tgcgcatgat ctagagctgc ctcgcgcgtt
     4981 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5041 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5101 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     5161 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     5221 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     5281 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5341 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5401 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5461 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5521 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5581 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5641 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5701 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5761 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5821 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5881 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5941 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6001 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6061 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6121 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6181 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6241 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6301 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6361 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6421 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6481 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6541 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6601 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6661 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6721 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6781 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6841 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6901 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6961 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7021 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7081 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7201 tattatcatg acattaacct at
//
