LOCUS       pVP33A.        7228 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-tetracysteine-TEV cassette 
            to an expressed protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP33A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7228)
  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 7228)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7228)
  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..7228
                     /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             1294..1317
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             1318..1335
                     /label=tetracysteine tag
                     /note="tetracysteine peptide that binds biarsenical
                     labeling reagents"
     CDS             1336..1356
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1385..1717
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        1736..1766
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1820..2476
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2489..2544
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2632..2652)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      2719..2813
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3583..3669
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(3728..3749)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3765..4844)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4845..4922)
                     /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    5114..5254
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5440..6028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6202..7059)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7060..7164)
                     /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 aacctggaag ttctgttcca ggggccctgt
     1321 tgcccaggtt gctgtgaaaa cctgtacttc caggcgatcg ccatggaata agtaaaggaa
     1381 tcacatggca caggttatca acacgtttga cggggttgcg gattatcttc agacatatca
     1441 taagctacct gataattaca ttacaaaatc agaagcacaa gccctcggct gggtggcatc
     1501 aaaagggaac cttgcagacg tcgctccggg gaaaagcatc ggcggagaca tcttctcaaa
     1561 cagggaaggc aaactcccgg gcaaaagcgg acgaacatgg cgtgaagcgg atattaacta
     1621 tacatcaggc ttcagaaatt cagaccggat tctttactca agcgactggc tgatttacaa
     1681 aacaacggac cattatcaga cctttacaaa aatcagataa ttaggcaccc caggctttac
     1741 actttatgct ttcggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
     1801 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     1861 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     1921 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     1981 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
     2041 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     2101 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     2161 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     2221 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     2281 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     2341 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     2401 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     2461 gtggcagggc ggggcgtaat gtttaaacga attcgagctc ggtacccggg gatcctctag
     2521 agtcgacctg caggcatgca agctgatccg gctgctaaca aagcccgaaa ggaagctgag
     2581 ttggctgctg ccaccgctga gcaataacta gcaaaaaact cgtttctcgt tcagctttct
     2641 tgtacaaagt ggtgatggcg cgcctgtagg acgtcgacgg taccatcgat acgcgttcga
     2701 agcttaatag ctgagctgga ctcctgttga tagatccagt aatgacctca gaactccatc
     2761 tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagct
     2821 agcttggcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     2881 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     2941 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     3001 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     3061 gctcatccgg aaatttcgta tggcaatgaa agacggtgag ctggtgatat ggggatagtg
     3121 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg
     3181 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg
     3241 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca
     3301 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg
     3361 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg
     3421 cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat
     3481 tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt
     3541 gcccttaaac gcctggggta atgactctct agcttgaggc atcaaataaa acgaaaggct
     3601 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     3661 aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa catgagaatt
     3721 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3781 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3841 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3901 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3961 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     4021 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     4081 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4141 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4201 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4261 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4321 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4381 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4441 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4501 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4561 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4621 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4681 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4741 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4801 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4861 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg
     4921 tcggaatttc gggcagcgtt gggtcctggc cacgggtgcg catgatctag agctgcctcg
     4981 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     5041 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     5101 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     5161 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     5221 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     5281 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5341 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5401 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5461 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5521 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5581 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5641 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5701 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5761 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5821 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5881 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5941 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6001 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6061 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6121 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6181 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6241 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6301 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6361 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6421 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6481 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6541 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6601 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6661 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6721 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6781 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6841 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6901 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6961 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7021 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7081 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7141 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     7201 aaccattatt atcatgacat taacctat
//
