LOCUS       V004512                 9568 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004512
VERSION     V004512
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9568)
  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 9568)
  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 9568)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9568)
  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..9568
                     /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(5282..5974)
                     /gene="SCEI"
                     /label="Intron-encoded endonuclease I-SceI"
                     /note="Intron-encoded endonuclease I-SceI from
                     Saccharomyces cerevisiae (strain ATCC 204508 / S288c).
                     Accession#: P03882"
     CDS             complement(6002..6997)
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             complement(7286..8377)
                     /label="LEU2"
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
     promoter        complement(8390..8794)
                     /label="LEU2 promoter"
     misc_feature    9065..9568
                     /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 cgtccttatt
     5281 atttcaggaa agtttcggag gagatagtgt tcggcagttt gtacatcatc tgcgggatca
     5341 ggtacggttt gatcaggttg tagaagatca ggtaagacat agaatcgatg tagatgatcg
     5401 gtttgttttt gttgattttt acgtaacagt tcagttggaa tttgttacgc agacccttaa
     5461 ccaggtattc tacttcttcg aaagtgaaag actgggtgtt cagtacgatc gatttgttgg
     5521 tagagttttt gttgtaatcc catttaccac catcatccat gaaccagtat gccagagaca
     5581 tcggggtcag gtagttttca accaggttgt tcgggatggt ttttttgttg ttaacgatga
     5641 acaggttagc cagtttgttg aaagcttggt gtttgaaagt ctgggcgccc caggtgatta
     5701 ccaggttacc caggtggtta acacgttgtt ttttgtgcgg cggggacagt acccactgat
     5761 cgtacagcag acatacgtgg tccatgtatg ctttgttttt ccactcgaac tgcatacagt
     5821 aggttttacc ttcatcacga gaacggatgt aagcatcacc caggatcaga ccgatacctg
     5881 cttcgaactg ttcgatgttc agttcgatca gctgggattt gtattctttc agcagtttag
     5941 agttcggacc caggttcatt acctggtttt tttgatgcat atgcgttctc ctacgcgttc
     6001 aggcgccggg ggcggtgtcc ggcggccccc agaggaactg cgccagttcc tccggatcgg
     6061 tgaagccgga gagatccagc ggggtctcct cgaacacctc gaagtcgtgc aggaaggtga
     6121 aggcgagcag ttcgcgggcg aagtcctcgg tccgcttcca ctgcgccccg tcgagcagcg
     6181 cggccaggat ctcgcggtcg ccccggaagg cgttgagatg cagttgcacc aggctgtagc
     6241 gggagtctcc cgcatagacg tcggtgaagt cgacgatccc ggtgacctcg gtcgcggcca
     6301 ggtccacgaa gatgttggtc ccgtgcaggt cgccgtggac gaaccggggt tcgcggccgg
     6361 ccagcagcgt gtccacgtcc ggcagccagt cctccaggcg gtccagcagc cggggcgaga
     6421 ggtagcccca cccgcggtgg tcctcgacgg tcgccgcgcg gcgttcccgc agcagttccg
     6481 ggaagacctc ggaatggggg gtgagcacgg tgttcccggt cagcggcacc ctgtgcagcc
     6541 ggccgagcac ccggccgagt tcgcgggcca gggcgagcag cgcgttccgg tcggtcgtgc
     6601 cgtccatcgc ggaccgccag gtggtgccgg tcatccggct catcaccagg tagggccacg
     6661 gccaggctcc ggtgccgggc cgcagctcgc cgcggacgag gaggcggggc accggcaccg
     6721 gggcgtccgc caggaccgcg tacgcctccg actccgacgc gaggctctcc ggaccgcacc
     6781 agtgctcgcc gaacagcttg atcaccgggc cgggctcgcc gaccagtacg gggttggtgc
     6841 tctcgccggg cacccgcagc accggcggca ccggcagccc gagctcctcc agggctcggc
     6901 gggccagcgg ctcccagaat tcctggtcgt tccgcaggct cgcgtaggaa tcatccgaat
     6961 caatacggtc gagaagtaac agggattctt gtgtcatatg aatcttactc ctttgttaaa
     7021 ttatttttgt ttaagcattt tgaatttgta tgcatcttag aacaatctca aagttccaag
     7081 ctcttgcaag tctagatatt gagtcaaaaa aaaaattttc aactcgagcc tcctttttct
     7141 ccttcttgat aaatgtatgt agattgcgta tatagtttcg tctaccctat gaacatattc
     7201 cattttgtaa tttcgtgtcg tttctattat gaatttcatt tataaagttt atgtacaaat
     7261 atcataaaaa aagagaatct ttttaagcaa ggattttctt aacttcttcg gcgacagcat
     7321 caccgacttc ggtggtactg ttggaaccac ctaaatcacc agttctgata cctgcatcca
     7381 aaaccttttt aactgcatct tcaatggcct taccttcttc aggcaagttc aatgacaatt
     7441 tcaacatcat tgcagcagac aagatagtgg cgatagggtc aaccttattc tttggcaaat
     7501 ctggagcaga accgtggcat ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag
     7561 aggccaagga cgcagatggc aacaaaccca aggaacctgg gataacggag gcttcatcgg
     7621 agatgatatc accaaacatg ttgctggtga ttataatacc atttaggtgg gttgggttct
     7681 taactaggat catggcggca gaatcaatca attgatgttg aaccttcaat gtagggaatt
     7741 cgttcttgat ggtttcctcc acagtttttc tccataatct tgaagaggcc aaaacattag
     7801 ctttatccaa ggaccaaata ggcaatggtg gctcatgttg tagggccatg aaagcggcca
     7861 ttcttgtgat tctttgcact tctggaacgg tgtattgttc actatcccaa gcgacaccat
     7921 caccatcgtc ttcctttctc ttaccaaagt aaatacctcc cactaattct ctgacaacaa
     7981 cgaagtcagt acctttagca aattgtggct tgattggaga taagtctaaa agagagtcgg
     8041 atgcaaagtt acatggtctt aagttggcgt acaattgaag ttctttacgg atttttagta
     8101 aaccttgttc aggtctaaca ctaccggtac cccatttagg accacccaca gcacctaaca
     8161 aaacggcatc aaccttcttg gaggcttcca gcgcctcatc tggaagtggg acacctgtag
     8221 catcgatagc agcaccacca attaaatgat tttcgaaatc gaacttgaca ttggaacgaa
     8281 catcagaaat agctttaaga accttaatgg cttcggctgt gatttcttga ccaacgtggt
     8341 cacctggcaa aacgacgatc ttcttagggg cagacatagg ggcagacatt agaatggtat
     8401 atccttgaaa tatatatata tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa
     8461 gacgattgct aaccacctat tggaaaaaac aataggtcct taaataatat tgtcaacttc
     8521 aagtattgtg atgcaagcat ttagtcatga acgcttctct attctatatg aaaagccggt
     8581 tccggcctct cacctttcct ttttctccca atttttcagt tgaaaaaggt atatgcgtca
     8641 ggcgacctct gaaattaaca aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg
     8701 cccctgtgtg ttctcgttat gttgaggaaa aaaataatgg ttgctaagag attcgaactc
     8761 ttgcatctta cgatacctga gtattcccac agttaactgc ggtcaagata tttcttgaat
     8821 caggcgcctt agaccgctcg gccaaacaac caattacttg ttgagaaata gagtataatt
     8881 atcctataaa tataacgttt ttgaacacac atgaacaagg aagtacagga caattgattt
     8941 tgaagagaat gtggattttg atgtaattgt tgggattcca tttttaataa ggcaataata
     9001 ttaggtatgt ggatatacta gaagttctcc tcgaccgtcg atagcttatc gatgggtcct
     9061 tttcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt
     9121 aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta
     9181 aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag
     9241 gtattaatgc cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg
     9301 attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct
     9361 aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt
     9421 ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa
     9481 acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga
     9541 ggcgcgtgta agttacaggc aagcgatc
//
