LOCUS       pVP33K.        7253 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial Flexi(R) Vector with a kanamycin resistance marker, for 
            appending an N-terminal 8xHis-MBP-HRV 3C-tetracysteine-TEV cassette 
            to an expressed protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP33K.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7253)
  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 7253)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7253)
  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..7253
                     /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             237..260
                     /label=8xHis
                     /note="8xHis affinity tag"
     CDS             267..1364
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1374..1397
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             1398..1415
                     /label=tetracysteine tag
                     /note="tetracysteine peptide that binds biarsenical
                     labeling reagents"
     CDS             1416..1436
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1465..1797
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        1816..1846
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1900..2556
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2569..2624
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2712..2732)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      2801..2895
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2939..3595
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     terminator      3663..3749
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3808..3829)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3845..4924)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4925..5002)
                     /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    5194..5334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5520..6108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6288..7079)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
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 atgggacatc
      241 accatcatca ccatcaccat gcatccaaaa tcgaagaagg taaactggta atctggatta
      301 acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg
      361 gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg
      421 caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc
      481 aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt
      541 ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag
      601 cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga
      661 tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc
      721 aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg
      781 aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc
      841 tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca
      901 tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat
      961 ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg
     1021 gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga
     1081 acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag
     1141 cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg
     1201 cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga
     1261 acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca
     1321 gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactttaatt aacctggaag
     1381 ttctgttcca ggggccctgt tgcccaggtt gctgtgaaaa cctgtacttc caggcgatcg
     1441 ccatggaata agtaaaggaa tcacatggca caggttatca acacgtttga cggggttgcg
     1501 gattatcttc agacatatca taagctacct gataattaca ttacaaaatc agaagcacaa
     1561 gccctcggct gggtggcatc aaaagggaac cttgcagacg tcgctccggg gaaaagcatc
     1621 ggcggagaca tcttctcaaa cagggaaggc aaactcccgg gcaaaagcgg acgaacatgg
     1681 cgtgaagcgg atattaacta tacatcaggc ttcagaaatt cagaccggat tctttactca
     1741 agcgactggc tgatttacaa aacaacggac cattatcaga cctttacaaa aatcagataa
     1801 ttaggcaccc caggctttac actttatgct ttcggctcgt ataatgtgtg gattttgagt
     1861 taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga
     1921 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca
     1981 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc
     2041 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg
     2101 aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt
     2161 gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt
     2221 gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac
     2281 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc
     2341 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc
     2401 gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg
     2461 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa
     2521 ttacaacagt actgcgatga gtggcagggc ggggcgtaat gtttaaacga attcgagctc
     2581 ggtacccggg gatcctctag agtcgacctg caggcatgca agctgatccg gctgctaaca
     2641 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcaaaaaact
     2701 cgtttctcgt tcagctttct tgtacaaagt ggtgatggcg cgcctgtagg acgtcgacgg
     2761 taccatcgat acgcgttcga agcttaatta gctgagcttg gactcctgtt gatagatcca
     2821 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     2881 tttattggtg agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat
     2941 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     3001 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     3061 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     3121 cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga
     3181 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     3241 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     3301 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     3361 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     3421 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     3481 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     3541 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt
     3601 tttttaaggc agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc
     3661 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     3721 cggtgaacgc tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata
     3781 agctgtcaaa catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     3841 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3901 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     3961 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4021 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4081 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4141 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4201 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4261 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4321 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4381 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4441 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4501 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4561 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4621 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4681 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4741 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     4801 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     4861 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     4921 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     4981 ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt gggtcctggc cacgggtgcg
     5041 catgatctag agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     5101 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     5161 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     5221 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     5281 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     5341 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5401 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5461 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5521 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5581 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5641 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5701 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5761 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5821 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5881 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5941 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6001 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6061 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6121 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6181 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6241 tcaatctaaa gtatatatga gtaaacttgg tctgacagcc taggtcagaa gaactcgtca
     6301 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     6361 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     6421 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     6481 ttttccacca tgatattcgg caagcaggca tcgccatgag tcacgacgag atcctcacca
     6541 tccggcatac gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     6601 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtgcgtgc tcgctcgatg
     6661 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     6721 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     6781 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     6841 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     6901 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     6961 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     7021 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaagcatg
     7081 cgaaacgacc gtcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt
     7141 ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc
     7201 ccagctggca attcttttga agctcacgct gccgcaagca ctcagggcgt acg
//
