LOCUS       pVP81K.        8538 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with a kanamycin resistance marker and the
            sacB gene, encoding an N-terminal MBP-TVMV-8xHis cassette plus TVMV 
            protease.
ACCESSION   .
VERSION     .
KEYWORDS    pVP81K
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8538)
  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 8538)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8538)
  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..8538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(20..106)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(142..849)
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
     promoter        complement(873..946)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        956..1000
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     protein_bind    1008..1024
                     /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             1047..1052
                     /note="ribosome binding site"
     CDS             1067..2164
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             2189..2209
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
     CDS             2210..2233
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             2485..3903
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        3992..4022
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             4076..4732
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    4745..4800
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(4885..4905)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      4992..5078
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(5137..5158)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5174..6253)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(6254..6331)
                     /label=lacI promoter
     misc_feature    6484..6624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6810..7398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7579..8370)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
ORIGIN
        1 cgtacgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
       61 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctctagcacg
      121 cgtatcattg gatctttatt agtccatgat ggcggcaaca gtttttttgg ccatgaagtc
      181 atcttccggc gcatcttcaa ccagggtaaa ggaaccccag ctgattttat ccgcgttgaa
      241 tttccagttt ttgcaccaac catccgcggc atcgagataa gtcgccacga atttttccgg
      301 aaattccacg aagtagttgc taccgttggt ggtatgagtc aggctgtgga tgcccagaat
      361 gttgccatca atgatggaaa ctagtgggct gccacactgg ccatctttag tggtgatcca
      421 gtgctgccag aaagaagtgt cttctttatg cacaatgtga gaggattcag acaccaggct
      481 ggacacgctt ttctgctgaa agttggtgga caccatgcac acacgatctt tgatggtcgg
      541 ctgacggaat ttcagtttct gcgggaacgg cgggaagtct ttagccattt tgataacgat
      601 aatgtcacgg ccttcaaccg gtttcatctg cagctgggta gagtttttga ctttgaattc
      661 accgtgcatg gttttgatgg tcagttcgcc attgttacga cgaaacagat gctggttggc
      721 aatgatatac gggccaaaac caatgccaaa cagacgttca ctatgaccat ccgaggagtt
      781 ttccagcagg catacgcaag cagagatcgg attaaaatcg cgcacgccct tcagcaaagc
      841 cttagacata tcttacctcc ttaaatcaat tcggtcagtg cgtcctgctg atgtgctcag
      901 tatctctatc actgataggg atgtcaatct ctatcactga tagggactcg agaaatcata
      961 aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg tgagcggata
     1021 acaatttcac acagaattca ttaaagagga gaaattaacc atgggtaaaa tcgaagaagg
     1081 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     1141 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     1201 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     1261 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     1321 ccaggacaag ctgtatccgt ttacctggga tgcggtacgt tacaacggca agctgattgc
     1381 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     1441 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     1501 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     1561 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     1621 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     1681 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     1741 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     1801 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     1861 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1921 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1981 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     2041 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     2101 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     2161 gactttaatt aacggcgacg gtgccgggga aaccgtgcgt ttccagtctc atcaccatca
     2221 tcaccatcac catgcatccg cgatcgcccg ttgcaatcca aacgagagtc taatagaatg
     2281 aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg
     2341 taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg
     2401 tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag
     2461 tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta
     2521 ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg
     2581 aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca tgatatgctg
     2641 caaatccctg aacagcaaaa aaatgaaaaa tatcaagttc ctgaattcga ttcgtccaca
     2701 attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac
     2761 gctgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat
     2821 cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct
     2881 attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat
     2941 gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac
     3001 ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg
     3061 acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag
     3121 gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc
     3181 gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa
     3241 gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa
     3301 ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa
     3361 gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct
     3421 ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt
     3481 gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa
     3541 tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac
     3601 gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac
     3661 aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca
     3721 cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac
     3781 agaggattct acgcagacaa acaatcaacg tttgcgccaa gcttcctgct gaacatcaaa
     3841 ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac
     3901 aaataaaaac gcaaaagaaa atgccgatat cctattggca ttttctttta tttcttatca
     3961 acataaaggt gaatagttag gcaccccagg ctttacactt tatgctttcg gctcgtataa
     4021 tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga
     4081 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     4141 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     4201 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     4261 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     4321 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     4381 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     4441 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     4501 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     4561 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     4621 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     4681 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaatgttt
     4741 aaacgaattc gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct
     4801 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     4861 taactagcaa actcgtttct cgttcagctt tcttgtacaa agtggtgatg gcgcgcctgt
     4921 aggacgtcga cggtaccatc gatacgcgtt cgaagcttcg cctggggtaa tgactctcta
     4981 gcttgaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     5041 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccctctaga ttacgtgcag
     5101 tcgatgataa gctgtcaaac atgagaattg tgcctaatga gtgagctaac ttacattaat
     5161 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5221 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5281 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5341 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5401 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5461 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5521 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5581 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5641 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5701 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5761 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5821 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5881 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5941 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6001 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6061 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6121 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6181 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6241 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6301 cgcgaaaggt tttgcgccat tcgatggtgt cggaacctag agctgcctcg cgcgtttcgg
     6361 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6421 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6481 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     6541 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     6601 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6661 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6721 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6781 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6841 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6901 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6961 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     7021 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7081 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7141 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7201 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     7261 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7321 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7381 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7441 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7501 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7561 tctgacagcc ctaggtcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     7621 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     7681 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     7741 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     7801 atcgccatga gtcacgacga gatcctcacc atccggcata cgcgccttga gcctggcgaa
     7861 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     7921 ggcttccatc cgagtgcgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     7981 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     8041 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     8101 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     8161 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     8221 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     8281 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     8341 acctgcgtgc aatccatctt gttcaagcat gcgaaacgac cgtcatcctg tctcttgatc
     8401 agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
     8461 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattcttttg aagctcacgc
     8521 tgccgcaagc actcaggg
//
