LOCUS       pVP91A.        6534 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with an ampicillin resistance marker, for 
            appending an N-terminal 8xHis tag to an expressed protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP91A.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6534)
  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 6534)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6534)
  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 sacB gene, insert an ORF between the SgfI site and the
            PmeI site or a downstream site.
FEATURES             Location/Qualifiers
     source          1..6534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        126..170
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    178..194
                     /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             217..222
                     /note="ribosome binding site"
     CDS             231..233
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             234..257
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             503..1921
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        2010..2040
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2094..2750
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2763..2818
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2903..2923)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      3010..3096
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3155..3176)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3192..4271)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4272..4349)
                     /label=lacI promoter
     misc_feature    4502..4642
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4828..5416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5595..6452)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6453..6524)
                     /label=AmpR promoter
ORIGIN
        1 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
       61 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcacctcg
      121 agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg
      181 tgagcggata acaatttcac acagaattca ttaaagagga gaaattaacc atgcatcacc
      241 atcatcacca tcaccatgcg atcgcccgtt gcaatccaaa cgagagtcta atagaatgag
      301 gtcgaaaagt aaatcgcgcg ggtttgttac tgataaagca ggcaagacct aaaatgtgta
      361 aagggcaaag tgtatacttt ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg
      421 cgtaactaac ttgccatctt caaacaggag ggctggaaga agcagaccgc taacacagta
      481 cataaaaaag gagacatgaa cgatgaacat caaaaagttt gcaaaacaag caacagtatt
      541 aacctttact accgcactgc tggcaggagg cgcaactcaa gcgtttgcga aagaaacgaa
      601 ccaaaagcca tataaggaaa catacggcat ttcccatatt acacgccatg atatgctgca
      661 aatccctgaa cagcaaaaaa atgaaaaata tcaagttcct gaattcgatt cgtccacaat
      721 taaaaatatc tcttctgcaa aaggcctgga cgtttgggac agctggccat tacaaaacgc
      781 tgacggcact gtcgcaaact atcacggcta ccacatcgtc tttgcattag ccggagatcc
      841 taaaaatgcg gatgacacat cgatttacat gttctatcaa aaagtcggcg aaacttctat
      901 tgacagctgg aaaaacgctg gccgcgtctt taaagacagc gacaaattcg atgcaaatga
      961 ttctatccta aaagaccaaa cacaagaatg gtcaggttca gccacattta catctgacgg
     1021 aaaaatccgt ttattctaca ctgatttctc cggtaaacat tacggcaaac aaacactgac
     1081 aactgcacaa gttaacgtat cagcatcaga cagctctttg aacatcaacg gtgtagagga
     1141 ttataaatca atctttgacg gtgacggaaa aacgtatcaa aatgtacagc agttcatcga
     1201 tgaaggcaac tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga
     1261 taaaggccac aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg
     1321 cgaagaatct ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga
     1381 aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct
     1441 cggtatgatt gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc
     1501 atctaacaca gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg
     1561 gtacctgttc actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga
     1621 tatttacatg cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa
     1681 aactggcctt gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca
     1741 cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag
     1801 aggattctac gcagacaaac aatcaacgtt tgcgccaagc ttcctgctga acatcaaagg
     1861 caagaaaaca tctgttgtca aagacagcat ccttgaacaa ggacaattaa cagttaacaa
     1921 ataaaaacgc aaaagaaaat gccgatatcc tattggcatt ttcttttatt tcttatcaac
     1981 ataaaggtga atagttaggc accccaggct ttacacttta tgctttcggc tcgtataatg
     2041 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     2101 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     2161 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     2221 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     2281 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     2341 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     2401 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     2461 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     2521 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     2581 tggacaactt cttcgccccc gttttcacga tgggcaaata ttatacgcaa ggcgacaagg
     2641 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     2701 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taatgtttaa
     2761 acgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggca tgcaagctga
     2821 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2881 actagcaaac tcgtttctcg ttcagctttc ttgtacaaag tggtgatggc gcgcctgtag
     2941 gacgtcgacg gtaccatcga tacgcgttcg aagcttcgcc tggggtaatg actctctagc
     3001 ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     3061 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cctctagatt acgtgcagtc
     3121 gatgataagc tgtcaaacat gagaattgtg cctaatgagt gagctaactt acattaattg
     3181 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3241 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     3301 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     3361 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     3421 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     3481 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     3541 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     3601 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     3661 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     3721 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     3781 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     3841 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     3901 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     3961 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     4021 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     4081 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     4141 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     4201 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     4261 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     4321 cgaaaggttt tgcgccattc gatggtgtcg gaacctagag ctgcctcgcg cgtttcggtg
     4381 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4441 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4501 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4561 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     4621 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4681 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4741 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4801 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4861 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4921 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4981 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5041 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5101 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5161 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5221 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5281 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5341 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5401 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5461 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5521 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5581 tgacagccta ggtaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5641 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5701 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5761 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5821 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5881 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5941 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6001 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6061 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6121 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6181 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6241 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6301 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6361 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6421 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6481 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattcg tacg
//
