LOCUS       V004502                 8843 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004502
VERSION     V004502
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8843)
  AUTHORS   Kalivoda EJ, Horzempa J, Stella NA, Sadaf A, Kowalski RP, Nau GJ,
            Shanks RM.
  TITLE     New vector tools with a hygromycin resistance marker for use with
            opportunistic pathogens
  JOURNAL   Mol. Biotechnol. 48 (1), 7-14 (2011)
   PUBMED   20972648
REFERENCE   2  (bases 1 to 8843)
  AUTHORS   Kalivoda EJ, Horzempa JE, Sadaf AA, Stella NA, Nau GJ, Shanks RM.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-DEC-2009) Ophthalmology, University of Pittsburgh, EEI
            1015, 203 Lothrop St, Pittsburgh, PA 15213, USA
REFERENCE   3  (bases 1 to 8843)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8843)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol.
            Biotechnol."; date: "2011"; volume: "48"; issue: "1"; pages: "7-14"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (19-DEC-2009) Ophthalmology, University of Pittsburgh, EEI 1015, 203
            Lothrop St, Pittsburgh, PA 15213, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            complement(399..508)
                     /direction=LEFT
                     /label="oriT"
                     /note="incP origin of transfer"
     terminator      complement(929..956)
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1048..1134)
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     1484..1500
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     misc_feature    complement(1504..1560)
                     /label="MCS"
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1570..1586)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(1594..1610)
                     /label="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)."
     promoter        complement(1618..1648)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1663..1684)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      1969..2357
                     /label="R6K gamma ori"
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2886..3533)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     CDS             5027..5041
                     /label="enterokinase site"
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(5277..6272)
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             complement(6561..7652)
                     /label="LEU2"
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
     promoter        complement(7665..8069)
                     /label="LEU2 promoter"
     misc_feature    8340..8843
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 tctaagaaac cattattatc atgacattaa cctataaaaa aggcctctcg agctagagtc
       61 gatcttcgcc agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc
      121 ggtgagccag agtttcagca ggccgcccag gcggcccagg tcgccattga tgcgggccag
      181 ctcgcggacg tgctcatagt ccacgacgcc cgtgattttg tagccctggc cgacggccag
      241 caggtaggcc gacaggctca tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc
      301 gtctggaagg cagtacacct tgataggtgg gctgcccttc ctggttggct tggtttcatc
      361 agccatccgc ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg
      421 attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc
      481 gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc
      541 tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa
      601 aatcgctata atgaccccga agcagggtta tgcagcggaa aagcgctgct tccctgctgt
      661 tttgtggaat atctaccgac tggaaacagg caaatgcagg aaattactga actgagggga
      721 caggcgagag acgatgccaa agagctacac cgacgagctg gccgagtggg ttgaatcccg
      781 cgcggccaag aagcgccggc gtgatgaggc tgcggttgcg ttcctggcgg tgagggcgga
      841 tgtcgatatg cgtaaggaga aaataccgca tcaggcgcat atttgaatgt atttagaaaa
      901 ataaacaaaa agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
      961 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     1021 cgcaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     1081 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     1141 ggcagttccc tactctcgca tggggagacc ccacactacc atcggcgcta cggcgtttca
     1201 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcaaattct
     1261 gttttatcag accgcttctg cgttctgatt taatctgtat caggctgaaa atcttctctc
     1321 atccgccaaa acagccaagc tcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     1381 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     1441 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     1501 gccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc
     1561 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1621 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1681 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1741 ttaatgcgcc ctacgggctt gccaaactgg aacaacactc accctatctc ggtctattct
     1801 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1861 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1921 ggggaaatgt gcgcggaacc agatccccac gatgcgtccg gcgtagagga tctgaagatc
     1981 agcagttcaa cctgttgata gtacgtacta agctctcatg tttcacgtac taagctctca
     2041 tgtttaacgt actaagctct catgtttaac gaactaaacc ctcatggcta acgtactaag
     2101 ctctcatggc taacgtacta agctctcatg tttcacgtac taagctctca tgtttgaaca
     2161 ataaaattaa tataaatcag caacttaaat agcctctaag gttttaagtt ttataagaaa
     2221 aaaaagaata tataaggctt ttaaagcttt taaggtttaa cggttgtgga caacaagcca
     2281 gggatgtaac gcactgagaa gcccttagag cctctcaaag caattttcag tgacacagga
     2341 acacttaacg gctgacagct gtaggttttt aggcataaaa ctatatgatt tacccctaaa
     2401 tctttaaaat gccccttaaa attcaaaata aaggcattta aaatttaaat atttcttgtg
     2461 ataaagtttg ttaaaaagga gtggttttat gactgttatg tggttatcga ttataggtat
     2521 gtggttttgt attggaatgg cattttttgc tatcaaggtt attaaaaata aaaattagac
     2581 cacgcattta tgccgagaaa atttattgtg cgttgagaag aacccttaac taaacttgca
     2641 gacgaatgtc ggcatagcgt gagctattaa gccgaccatt cgacaagttt tgggattgtt
     2701 aagggttccg aggctcaacg tcaataaagc aattggaata aagaagcgaa aaaggagaag
     2761 tcggttcaga aaaagaagga tatggatctg gagctgtaat ataaaaacct tcttcaacta
     2821 acggggcagg ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg cattatctca
     2881 tattataaaa gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat
     2941 cctgcatgat aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt
     3001 gaattacctt tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta
     3061 ttattgatat ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa
     3121 gcattttcag gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca
     3181 tcagaaaggt ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca
     3241 gagaatgttt tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa
     3301 cctaactctc cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt
     3361 gtcactaaga aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat
     3421 aaaacactaa tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga
     3481 ttaaatatct cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat
     3541 gcctcctaaa tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta
     3601 gaatcaatcc ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat
     3661 cccactttat ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga
     3721 ccacattaaa aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc
     3781 cttttctttc ttatcttgat aataagggta actattgccg gcgaggctag ttacccttaa
     3841 gttattggta tgactggttt taagcgcaaa aaaagttgct ttttcgtacc tattaatgta
     3901 tcgttttaaa tgattagtaa aaaacataca tagaaagggg aaaaagcaac tttttttatt
     3961 gtcatagttt gtgaaaacta agttgttttt atgtgttata acatggaaaa gtatactgag
     4021 aaaaaacaaa gaaatcaagt atttcagaaa tttattaaac gtcatattgg agagaatcaa
     4081 atggatttag ttgaagattg caatacattt ctgtcttttg tagctgataa aactttagaa
     4141 aaacagaaat tatataaagc taattcttgt aaaaatcgat tttgtcctgt ctgtgcttgg
     4201 agaaaagcta gaaaagatgc attgggttta tctttgatga tgcaatatat taagcagcaa
     4261 gagaaaaagg agtttatctt tttaactttg actacaccta atgtaatgag tgatgaatta
     4321 gaaaatgaaa taaaacgtta taataattct tttagaaaac ttataaagag aaaaaaagta
     4381 ggtagtgtta taaagggata tgttcgtaag ttagagatta catataataa aaaaagagat
     4441 gattataatc ctcattttca tgtgttaatt gcagtaaata aatcgtattt cacagataaa
     4501 agatattata ttagccaaca agaatggtta gatttatggc gtgatgtaac gggcatttca
     4561 gaaataacac aagttcaagt tcaaaaaata agacaaaata ataataaaga attatatgaa
     4621 atggctaagt attctggtaa agatagtgat tatttaataa ataaatcaaa aagtctttga
     4681 tgcattttat aaatcactta aaggtaaaca ggtattagtt tattcaggat tatttaaaga
     4741 ggctaaaaag aaattaaaaa atggggattt agattactta aaagaaattg atccaaccgt
     4801 aatatatcta tcaaattttt tatatttgga aacaaaaaga gtatttagct agtgaacttt
     4861 atgacttaac agaacaagaa aaaagagaaa ttaatcacaa aatgatagac gaaatcgagg
     4921 aagaacaata acaaaatata agtgctaaca gctgacctcc cgataacacc atgtagttat
     4981 tgggaggtca gcgtgaatta tgcacgagta ttttaaaagt tattgtgatg acgacgataa
     5041 acgattatca aaagtataat gttaaaatgc tttattatac taacgttata taaacattat
     5101 actttcgtta tacaaatttt aaccctgtta ggaactataa aaaatcatga aaattttaat
     5161 ttgcatgtaa ctgggcagtg tcttaaaaaa tcgacactga atttgctcaa atttttgttt
     5221 gtagaattag aatatattta tttggctcat atttgctttt taaaagcacg cgttcaggcg
     5281 ccgggggcgg tgtccggcgg cccccagagg aactgcgcca gttcctccgg atcggtgaag
     5341 ccggagagat ccagcggggt ctcctcgaac acctcgaagt cgtgcaggaa ggtgaaggcg
     5401 agcagttcgc gggcgaagtc ctcggtccgc ttccactgcg ccccgtcgag cagcgcggcc
     5461 aggatctcgc ggtcgccccg gaaggcgttg agatgcagtt gcaccaggct gtagcgggag
     5521 tctcccgcat agacgtcggt gaagtcgacg atcccggtga cctcggtcgc ggccaggtcc
     5581 acgaagatgt tggtcccgtg caggtcgccg tggacgaacc ggggttcgcg gccggccagc
     5641 agcgtgtcca cgtccggcag ccagtcctcc aggcggtcca gcagccgggg cgagaggtag
     5701 ccccacccgc ggtggtcctc gacggtcgcc gcgcggcgtt cccgcagcag ttccgggaag
     5761 acctcggaat ggggggtgag cacggtgttc ccggtcagcg gcaccctgtg cagccggccg
     5821 agcacccggc cgagttcgcg ggccagggcg agcagcgcgt tccggtcggt cgtgccgtcc
     5881 atcgcggacc gccaggtggt gccggtcatc cggctcatca ccaggtaggg ccacggccag
     5941 gctccggtgc cgggccgcag ctcgccgcgg acgaggaggc ggggcaccgg caccggggcg
     6001 tccgccagga ccgcgtacgc ctccgactcc gacgcgaggc tctccggacc gcaccagtgc
     6061 tcgccgaaca gcttgatcac cgggccgggc tcgccgacca gtacggggtt ggtgctctcg
     6121 ccgggcaccc gcagcaccgg cggcaccggc agcccgagct cctccagggc tcggcgggcc
     6181 agcggctccc agaattcctg gtcgttccgc aggctcgcgt aggaatcatc cgaatcaata
     6241 cggtcgagaa gtaacaggga ttcttgtgtc atatgaatct tactcctttg ttaaattatt
     6301 tttgtttaag cattttgaat ttgtatgcat cttagaacaa tctcaaagtt ccaagctctt
     6361 gcaagtctag atattgagtc aaaaaaaaaa ttttcaactc gagcctcctt tttctccttc
     6421 ttgataaatg tatgtagatt gcgtatatag tttcgtctac cctatgaaca tattccattt
     6481 tgtaatttcg tgtcgtttct attatgaatt tcatttataa agtttatgta caaatatcat
     6541 aaaaaaagag aatcttttta agcaaggatt ttcttaactt cttcggcgac agcatcaccg
     6601 acttcggtgg tactgttgga accacctaaa tcaccagttc tgatacctgc atccaaaacc
     6661 tttttaactg catcttcaat ggccttacct tcttcaggca agttcaatga caatttcaac
     6721 atcattgcag cagacaagat agtggcgata gggtcaacct tattctttgg caaatctgga
     6781 gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt tcttgtctgg caaagaggcc
     6841 aaggacgcag atggcaacaa acccaaggaa cctgggataa cggaggcttc atcggagatg
     6901 atatcaccaa acatgttgct ggtgattata ataccattta ggtgggttgg gttcttaact
     6961 aggatcatgg cggcagaatc aatcaattga tgttgaacct tcaatgtagg gaattcgttc
     7021 ttgatggttt cctccacagt ttttctccat aatcttgaag aggccaaaac attagcttta
     7081 tccaaggacc aaataggcaa tggtggctca tgttgtaggg ccatgaaagc ggccattctt
     7141 gtgattcttt gcacttctgg aacggtgtat tgttcactat cccaagcgac accatcacca
     7201 tcgtcttcct ttctcttacc aaagtaaata cctcccacta attctctgac aacaacgaag
     7261 tcagtacctt tagcaaattg tggcttgatt ggagataagt ctaaaagaga gtcggatgca
     7321 aagttacatg gtcttaagtt ggcgtacaat tgaagttctt tacggatttt tagtaaacct
     7381 tgttcaggtc taacactacc ggtaccccat ttaggaccac ccacagcacc taacaaaacg
     7441 gcatcaacct tcttggaggc ttccagcgcc tcatctggaa gtgggacacc tgtagcatcg
     7501 atagcagcac caccaattaa atgattttcg aaatcgaact tgacattgga acgaacatca
     7561 gaaatagctt taagaacctt aatggcttcg gctgtgattt cttgaccaac gtggtcacct
     7621 ggcaaaacga cgatcttctt aggggcagac ataggggcag acattagaat ggtatatcct
     7681 tgaaatatat atatatattg ctgaaatgta aaaggtaaga aaagttagaa agtaagacga
     7741 ttgctaacca cctattggaa aaaacaatag gtccttaaat aatattgtca acttcaagta
     7801 ttgtgatgca agcatttagt catgaacgct tctctattct atatgaaaag ccggttccgg
     7861 cctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga
     7921 cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct
     7981 gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca
     8041 tcttacgata cctgagtatt cccacagtta actgcggtca agatatttct tgaatcaggc
     8101 gccttagacc gctcggccaa acaaccaatt acttgttgag aaatagagta taattatcct
     8161 ataaatataa cgtttttgaa cacacatgaa caaggaagta caggacaatt gattttgaag
     8221 agaatgtgga ttttgatgta attgttggga ttccattttt aataaggcaa taatattagg
     8281 tatgtggata tactagaagt tctcctcgac cgtcgatagc ttatcgatgg gtccttttca
     8341 tcacgtgcta taaaaataat tataatttaa attttttaat ataaatatat aaattaaaaa
     8401 tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg ttttccgaag atgtaaaaga
     8461 ctctaggggg atcgccaaca aatactacct tttatcttgc tcttcctgct ctcaggtatt
     8521 aatgccgaat tgtttcatct tgtctgtgta gaagaccaca cacgaaaatc ctgtgatttt
     8581 acattttact tatcgttaat cgaatgtata tctatttaat ctgcttttct tgtctaataa
     8641 atatatatgt aaagtacgct ttttgttgaa attttttaaa cctttgttta tttttttttc
     8701 ttcattccgt aactcttcta ccttctttat ttactttcta aaatccaaat acaaaacata
     8761 aaaataaata aacacagagt aaattcccaa attattccat cattaaaaga tacgaggcgc
     8821 gtgtaagtta caggcaagcg atc
//
