LOCUS       pVP68K.        6353 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 cassette to an expressed 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVP68K
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6353)
  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 6353)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6353)
  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..6353
                     /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             1389..1412
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             1450..1782
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
     promoter        1801..1831
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1885..2541
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     misc_feature    2554..2609
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     protein_bind    complement(2694..2714)
                     /label=attB2
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     terminator      2801..2887
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(2946..2967)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2983..4062)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4063..4140)
                     /label=lacI promoter
     misc_feature    4293..4433
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4619..5207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5388..6179)
                     /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 aacggcgacg
     1381 gtgccgggct ggaagttctg ttccaggggc ccgcgatcgc ggccgcgttg gaataagtaa
     1441 aggaatcaca tggcacaggt tatcaacacg tttgacgggg ttgcggatta tcttcagaca
     1501 tatcataagc tacctgataa ttacattaca aaatcagaag cacaagccct cggctgggtg
     1561 gcatcaaaag ggaaccttgc agacgtcgct ccggggaaaa gcatcggcgg agacatcttc
     1621 tcaaacaggg aaggcaaact cccgggcaaa agcggacgaa catggcgtga agcggatatt
     1681 aactatacat caggcttcag aaattcagac cggattcttt actcaagcga ctggctgatt
     1741 tacaaaacaa cggaccatta tcagaccttt acaaaaatca gataattagg caccccaggc
     1801 tttacacttt atgctttcgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga
     1861 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     1921 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     1981 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     2041 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     2101 ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     2161 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     2221 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
     2281 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     2341 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacg
     2401 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     2461 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     2521 gatgagtggc agggcggggc gtaatgttta aacgaattcg agctcggtac ccggggatcc
     2581 tctagagtcg acctgcaggc atgcaagctg atccggctgc taacaaagcc cgaaaggaag
     2641 ctgagttggc tgctgccacc gctgagcaat aactagcaaa ctcgtttctc gttcagcttt
     2701 cttgtacaaa gtggtgatgg cgcgcctgta ggacgtcgac ggtaccatcg atacgcgttc
     2761 gaagcttcgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     2821 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2881 gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt
     2941 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3001 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3061 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     3121 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3181 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     3241 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3301 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     3361 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     3421 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     3481 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3541 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3601 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3661 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     3721 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3781 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3841 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3901 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3961 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4021 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4081 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     4141 ggaacctaga gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4201 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4261 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     4321 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4381 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     4441 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4501 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4561 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4621 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4681 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4741 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4801 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4861 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4921 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4981 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5041 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5101 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5161 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5221 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5281 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5341 caatctaaag tatatatgag taaacttggt ctgacagccc taggtcagaa gaactcgtca
     5401 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     5461 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     5521 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     5581 ttttccacca tgatattcgg caagcaggca tcgccatgag tcacgacgag atcctcacca
     5641 tccggcatac gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     5701 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtgcgtgc tcgctcgatg
     5761 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     5821 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     5881 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     5941 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     6001 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     6061 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     6121 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaagcatg
     6181 cgaaacgacc gtcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt
     6241 ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc
     6301 ccagctggca attcttttga agctcacgct gccgcaagca ctcagggcgt acg
//
