LOCUS       V004513                 7076 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004513
VERSION     V004513
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7076)
  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 7076)
  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 7076)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7076)
  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..7076
                     /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      1834..2603
                     /label="pBBR1 oriV"
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1
                     Rep protein for replication"
     CDS             2604..3263
                     /label="pBBR1 Rep"
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     CDS             complement(3510..4505)
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             complement(4794..5885)
                     /label="LEU2"
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
     promoter        complement(5898..6302)
                     /label="LEU2 promoter"
     misc_feature    6573..7076
                     /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 gctctccggg cttcgccctg cgcggtcgct gcgctccctt
     1801 gccagcccgt ggatatgtgg acgatggccg cgagcggcca ccggctggct cgcttcgctc
     1861 ggcccgtgga caaccctgct ggacaagctg atggacaggc tgcgcctgcc cacgagcttg
     1921 accacaggga ttgcccaccg gctacccagc cttcgaccac atacccaccg gctccgactg
     1981 cgcggcctgc ggccttgccc catcaatttt tttaattttc tctggggaaa agcctccggc
     2041 ctgcggcctg cgcgcttcgc ttgccggttg gacaccaagt ggaaggcggg tcaaggctcg
     2101 cgcagcgacc gcgcagcggc ttggccttga cgcgcctgga acgacccaag cctatgcgag
     2161 tgggggcagt cgaaggcgaa gcccgcccgc ctgccccccg agcctcacgg cggcgagtgc
     2221 gggggttcca agggggcagc gccaccttgg gcaaggccga aggccgcgca gtcgatcaac
     2281 aagccccgga ggggccactt tttgccggag ggggagccgc gccgaaggcg tgggggaacc
     2341 ccgcaggggt gcccttcttt gggcaccaaa gaactagata tagggcgaaa tgcgaaagac
     2401 ttaaaaatca acaacttaaa aaaggggggt acgcaacagc tcattgcggc accccccgca
     2461 atagctcatt gcgtaggtta aagaaaatct gtaattgact gccactttta cgcaacgcat
     2521 aattgttgtc gcgctgccga aaagttgcag ctgattgcgc atggtgccgc aaccgtgcgg
     2581 caccctaccg catggagata agcatggcca cgcagtccag agaaatcggc attcaagcca
     2641 agaacaagcc cggtcactgg gtgcaaacgg aacgcaaagc gcatgaggcg tgggccgggc
     2701 ttattgcgag gaaacccacg gcggcaatgc tgctgcatca cctcgtggcg cagatgggcc
     2761 accagaacgc cgtggtggtc agccagaaga cactttccaa gctcatcgga cgttctttgc
     2821 ggacggtcca atacgcagtc aaggacttgg tggccgagcg ctggatctcc gtcgtgaagc
     2881 tcaacggccc cggcaccgtg tcggcctacg tggtcaatga ccgcgtggcg tggggccagc
     2941 cccgcgacca gttgcgcctg tcggtgttca gtgccgccgt ggtggttgat cacgacgacc
     3001 aggacgaatc gctgttgggg catggcgacc tgcgccgcat cccgaccctg tatccgggcg
     3061 agcagcaact accgaccggc cccggcgagg agccgcccag ccagcccggc attccgggca
     3121 tggaaccaga cctgccagcc ttgaccgaaa cggaggaatg ggaacggcgc gggcagcagc
     3181 gcctgccgat gcccgatgag ccgtgttttc tggacgatgg cgagccgttg gagccgccga
     3241 cacgggtcac gctgccgcgc cggtagcact tgggttgcgc agcaacccgt aagtgcgctg
     3301 ttccagacta tcggctgtag ccgcctcgcc gccctatacc ttgtctgcct ccccgcgttg
     3361 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagcctttta cgagtttttg
     3421 gtgcaatcaa tctaaagtat atatgagtaa acttggtctg acaagagagt gcaccataat
     3481 cggcattttc ttttgcgttt acgcgttcag gcgccggggg cggtgtccgg cggcccccag
     3541 aggaactgcg ccagttcctc cggatcggtg aagccggaga gatccagcgg ggtctcctcg
     3601 aacacctcga agtcgtgcag gaaggtgaag gcgagcagtt cgcgggcgaa gtcctcggtc
     3661 cgcttccact gcgccccgtc gagcagcgcg gccaggatct cgcggtcgcc ccggaaggcg
     3721 ttgagatgca gttgcaccag gctgtagcgg gagtctcccg catagacgtc ggtgaagtcg
     3781 acgatcccgg tgacctcggt cgcggccagg tccacgaaga tgttggtccc gtgcaggtcg
     3841 ccgtggacga accggggttc gcggccggcc agcagcgtgt ccacgtccgg cagccagtcc
     3901 tccaggcggt ccagcagccg gggcgagagg tagccccacc cgcggtggtc ctcgacggtc
     3961 gccgcgcggc gttcccgcag cagttccggg aagacctcgg aatggggggt gagcacggtg
     4021 ttcccggtca gcggcaccct gtgcagccgg ccgagcaccc ggccgagttc gcgggccagg
     4081 gcgagcagcg cgttccggtc ggtcgtgccg tccatcgcgg accgccaggt ggtgccggtc
     4141 atccggctca tcaccaggta gggccacggc caggctccgg tgccgggccg cagctcgccg
     4201 cggccgagga ggcggggcac cggcaccggg gcgtccgcca ggaccgcgta cgcctccgac
     4261 tccgacgcga ggctctccgg accgcaccag tgctcgccga acagcttgat caccgggtcg
     4321 ggctcgccga ccagtacggg gttggtgctc tcgccgggca cccgcagcac cggcggcacc
     4381 ggcagcccga gctcctccag ggctcggcgg gccagcggct cccagaattc ctggtcgttc
     4441 cgcaggctcg cgtaggaatc atccgaatca atacggtcga gaagtaacag ggattcttgt
     4501 gtcatatgaa tcttactcct ttgttaaatt atttttgttt aagcattttg aatttgtatg
     4561 catcttagaa caatctcaaa gttccaagct cttgcaagtc tagatattga gtcaaaaaaa
     4621 aaattttcaa ctcgagcctc ctttttctcc ttcttgataa atgtatgtag attgcgtata
     4681 tagtttcgtc taccctatga acatattcca ttttgtaatt tcgtgtcgtt tctattatga
     4741 atttcattta taaagtttat gtacaaatat cataaaaaaa gagaatcttt ttaagcaagg
     4801 attttcttaa cttcttcggc gacagcatca ccgacttcgg tggtactgtt ggaaccacct
     4861 aaatcaccag ttctgatacc tgcatccaaa acctttttaa ctgcatcttc aatggcctta
     4921 ccttcttcag gcaagttcaa tgacaatttc aacatcattg cagcagacaa gatagtggcg
     4981 atagggtcaa ccttattctt tggcaaatct ggagcagaac cgtggcatgg ttcgtacaaa
     5041 ccaaatgcgg tgttcttgtc tggcaaagag gccaaggacg cagatggcaa caaacccaag
     5101 gaacctggga taacggaggc ttcatcggag atgatatcac caaacatgtt gctggtgatt
     5161 ataataccat ttaggtgggt tgggttctta actaggatca tggcggcaga atcaatcaat
     5221 tgatgttgaa ccttcaatgt agggaattcg ttcttgatgg tttcctccac agtttttctc
     5281 cataatcttg aagaggccaa aacattagct ttatccaagg accaaatagg caatggtggc
     5341 tcatgttgta gggccatgaa agcggccatt cttgtgattc tttgcacttc tggaacggtg
     5401 tattgttcac tatcccaagc gacaccatca ccatcgtctt cctttctctt accaaagtaa
     5461 atacctccca ctaattctct gacaacaacg aagtcagtac ctttagcaaa ttgtggcttg
     5521 attggagata agtctaaaag agagtcggat gcaaagttac atggtcttaa gttggcgtac
     5581 aattgaagtt ctttacggat ttttagtaaa ccttgttcag gtctaacact accggtaccc
     5641 catttaggac cacccacagc acctaacaaa acggcatcaa ccttcttgga ggcttccagc
     5701 gcctcatctg gaagtgggac acctgtagca tcgatagcag caccaccaat taaatgattt
     5761 tcgaaatcga acttgacatt ggaacgaaca tcagaaatag ctttaagaac cttaatggct
     5821 tcggctgtga tttcttgacc aacgtggtca cctggcaaaa cgacgatctt cttaggggca
     5881 gacatagggg cagacattag aatggtatat ccttgaaata tatatatata ttgctgaaat
     5941 gtaaaaggta agaaaagtta gaaagtaaga cgattgctaa ccacctattg gaaaaaacaa
     6001 taggtcctta aataatattg tcaacttcaa gtattgtgat gcaagcattt agtcatgaac
     6061 gcttctctat tctatatgaa aagccggttc cggcctctca cctttccttt ttctcccaat
     6121 ttttcagttg aaaaaggtat atgcgtcagg cgacctctga aattaacaaa aaatttccag
     6181 tcatcgaatt tgattctgtg cgatagcgcc cctgtgtgtt ctcgttatgt tgaggaaaaa
     6241 aataatggtt gctaagagat tcgaactctt gcatcttacg atacctgagt attcccacag
     6301 ttaactgcgg tcaagatatt tcttgaatca ggcgccttag accgctcggc caaacaacca
     6361 attacttgtt gagaaataga gtataattat cctataaata taacgttttt gaacacacat
     6421 gaacaaggaa gtacaggaca attgattttg aagagaatgt ggattttgat gtaattgttg
     6481 ggattccatt tttaataagg caataatatt aggtatgtgg atatactaga agttctcctc
     6541 gaccgtcgat agcttatcga tgggtccttt tcatcacgtg ctataaaaat aattataatt
     6601 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
     6661 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
     6721 ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
     6781 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
     6841 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
     6901 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
     6961 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
     7021 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatc
//
