LOCUS       pVP65K.        7174 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with a kanamycin resistance marker and the
            barnase gene, encoding an N-terminal MBP-TVMV-8xHis cassette plus 
            TVMV protease.
ACCESSION   .
VERSION     .
KEYWORDS    pVP65K.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  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 7174)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7174)
  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..7174
                     /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             2277..2609
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        2628..2658
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2712..3368
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    3381..3436
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(3521..3541)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      3628..3714
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3773..3794)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3810..4889)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4890..4967)
                     /label=lacI promoter
     misc_feature    5120..5260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5446..6034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6215..7006)
                     /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 cgatcgcggc cgcgttggaa taagtaaagg aatcacatgg
     2281 cacaggttat caacacgttt gacggggttg cggattatct tcagacatat cataagctac
     2341 ctgataatta cattacaaaa tcagaagcac aagccctcgg ctgggtggca tcaaaaggga
     2401 accttgcaga cgtcgctccg gggaaaagca tcggcggaga catcttctca aacaaggaag
     2461 gcaaactccc gggcaaaagc ggacgaacat ggcgtgaagc ggatattaac tatacatcag
     2521 gcttcagaaa ttcagaccgg attctttact caagcgactg gctgatttac aaaacaacgg
     2581 accattatca gacctttaca aaaatcagat aattaggcac cccaggcttt acactttatg
     2641 ctttcggctc gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta
     2701 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     2761 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     2821 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     2881 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
     2941 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     3001 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     3061 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     3121 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     3181 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcacgatg ggcaaatatt
     3241 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     3301 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     3361 gcggggcgta atgtttaaac gaattcgagc tcggtacccg gggatcctct agagtcgacc
     3421 tgcaggcatg caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3481 tgccaccgct gagcaataac tagcaaactc gtttctcgtt cagctttctt gtacaaagtg
     3541 gtgatggcgc gcctgtagga cgtcgacggt accatcgata cgcgttcgaa gcttcgcctg
     3601 gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     3661 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccc
     3721 tctagattac gtgcagtcga tgataagctg tcaaacatga gaattgtgcc taatgagtga
     3781 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3841 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3901 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3961 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4021 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4081 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4141 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4201 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4261 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4321 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4381 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4441 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4501 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4561 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4621 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4681 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4741 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4801 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4861 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4921 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtcgga acctagagct
     4981 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5041 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5101 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     5161 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     5221 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     5281 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5341 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5401 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5461 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5521 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5581 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5641 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5701 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5761 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5821 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5881 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5941 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6001 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6061 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6121 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6181 atatgagtaa acttggtctg acagccctag gtcagaagaa ctcgtcaaga aggcgataga
     6241 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     6301 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     6361 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     6421 tattcggcaa gcaggcatcg ccatgagtca cgacgagatc ctcaccatcc ggcatacgcg
     6481 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     6541 cctgatcgac aagaccggct tccatccgag tgcgtgctcg ctcgatgcga tgtttcgctt
     6601 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     6661 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     6721 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     6781 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     6841 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     6901 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     6961 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aagcatgcga aacgaccgtc
     7021 atcctgtctc ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag
     7081 ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt
     7141 cttttgaagc tcacgctgcc gcaagcactc aggg
//
