LOCUS       V004503                 8677 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004503
VERSION     V004503
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8677)
  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 8677)
  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 8677)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8677)
  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..8677
                     /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(3669..5087)
                     /label="SacB"
                     /note="secreted levansucrase that renders bacterial growth
                     sensitive to sucrose"
     CDS             complement(5111..6106)
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             complement(6395..7486)
                     /label="LEU2"
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
     promoter        complement(7499..7903)
                     /label="LEU2 promoter"
     misc_feature    8174..8677
                     /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 acgcgtggag acggtcacag cttgtctgta agcggatgcc
     3541 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     3601 aactatgcgg catcagagca gattgtactg agagtgcacc ataatcggca ttttcttttg
     3661 cgtttttatt tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat
     3721 gttttcttgc ctttgatgtt cagcaggaag ctaggcgcaa acgttgattg tttgtctgcg
     3781 tagaatcctc tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt
     3841 acagcgaagt gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca
     3901 aggccagttt tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc
     3961 atgtaaatat cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg
     4021 aacagatacc atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact
     4081 gtgttagatg caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca
     4141 atcataccga gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt
     4201 ttttgacttt cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa
     4261 gattcttcgc cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg
     4321 tggcctttat cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag
     4381 ttgccttcat cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt
     4441 gatttataat cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact
     4501 tgtgcagttg tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa
     4561 cggatttttc cgtcagatgt aaatgtggct gaacctgacc attcttgtgt ttggtctttt
     4621 aggatagaat catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc
     4681 cagctgtcaa tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc
     4741 gcatttttag gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca
     4801 gtgccgtcag cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag
     4861 atatttttaa ttgtggacga atcgaactca ggaacttgat atttttcatt tttttgctgt
     4921 tcagggattt gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat
     4981 ggcttttggt tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta
     5041 gtaaaggtta atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc
     5101 tacgcgttca ggcgccgggg gcggtgtccg gcggccccca gaggaactgc gccagttcct
     5161 ccggatcggt gaagccggag agatccagcg gggtctcctc gaacacctcg aagtcgtgca
     5221 ggaaggtgaa ggcgagcagt tcgcgggcga agtcctcggt ccgcttccac tgcgccccgt
     5281 cgagcagcgc ggccaggatc tcgcggtcgc cccggaaggc gttgagatgc agttgcacca
     5341 ggctgtagcg ggagtctccc gcatagacgt cggtgaagtc gacgatcccg gtgacctcgg
     5401 tcgcggccag gtccacgaag atgttggtcc cgtgcaggtc gccgtggacg aaccggggtt
     5461 cgcggccggc cagcagcgtg tccacgtccg gcagccagtc ctccaggcgg tccagcagcc
     5521 ggggcgagag gtagccccac ccgcggtggt cctcgacggt cgccgcgcgg cgttcccgca
     5581 gcagttccgg gaagacctcg gaatgggggg tgagcacggt gttcccggtc agcggcaccc
     5641 tgtgcagccg gccgagcacc cggccgagtt cgcgggccag ggcgagcagc gcgttccggt
     5701 cggtcgtgcc gtccatcgcg gaccgccagg tggtgccggt catccggctc atcaccaggt
     5761 agggccacgg ccaggctccg gtgccgggcc gcagctcgcc gcggccgagg aggcggggca
     5821 ccggcaccgg ggcgtccgcc aggaccgcgt acgcctccga ctccgacgcg aggctctccg
     5881 gaccgcacca gtgctcgccg aacagcttga tcaccgggtc gggctcgccg accagtacgg
     5941 ggttggtgct ctcgccgggc acccgcagca ccggcggcac cggcagcccg agctcctcca
     6001 gggctcggcg ggccagcggc tcccagaatt cctggtcgtt ccgcaggctc gcgtaggaat
     6061 catccgaatc aatacggtcg agaagtaaca gggattcttg tgtcatatga atcttactcc
     6121 tttgttaaat tatttttgtt taagcatttt gaatttgtat gcatcttaga acaatctcaa
     6181 agttccaagc tcttgcaagt ctagatattg agtcaaaaaa aaaattttca actcgagcct
     6241 cctttttctc cttcttgata aatgtatgta gattgcgtat atagtttcgt ctaccctatg
     6301 aacatattcc attttgtaat ttcgtgtcgt ttctattatg aatttcattt ataaagttta
     6361 tgtacaaata tcataaaaaa agagaatctt tttaagcaag gattttctta acttcttcgg
     6421 cgacagcatc accgacttcg gtggtactgt tggaaccacc taaatcacca gttctgatac
     6481 ctgcatccaa aaccttttta actgcatctt caatggcctt accttcttca ggcaagttca
     6541 atgacaattt caacatcatt gcagcagaca agatagtggc gatagggtca accttattct
     6601 ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt
     6661 ctggcaaaga ggccaaggac gcagatggca acaaacccaa ggaacctggg ataacggagg
     6721 cttcatcgga gatgatatca ccaaacatgt tgctggtgat tataatacca tttaggtggg
     6781 ttgggttctt aactaggatc atggcggcag aatcaatcaa ttgatgttga accttcaatg
     6841 tagggaattc gttcttgatg gtttcctcca cagtttttct ccataatctt gaagaggcca
     6901 aaacattagc tttatccaag gaccaaatag gcaatggtgg ctcatgttgt agggccatga
     6961 aagcggccat tcttgtgatt ctttgcactt ctggaacggt gtattgttca ctatcccaag
     7021 cgacaccatc accatcgtct tcctttctct taccaaagta aatacctccc actaattctc
     7081 tgacaacaac gaagtcagta cctttagcaa attgtggctt gattggagat aagtctaaaa
     7141 gagagtcgga tgcaaagtta catggtctta agttggcgta caattgaagt tctttacgga
     7201 tttttagtaa accttgttca ggtctaacac taccggtacc ccatttagga ccacccacag
     7261 cacctaacaa aacggcatca accttcttgg aggcttccag cgcctcatct ggaagtggga
     7321 cacctgtagc atcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat
     7381 tggaacgaac atcagaaata gctttaagaa ccttaatggc ttcggctgtg atttcttgac
     7441 caacgtggtc acctggcaaa acgacgatct tcttaggggc agacataggg gcagacatta
     7501 gaatggtata tccttgaaat atatatatat attgctgaaa tgtaaaaggt aagaaaagtt
     7561 agaaagtaag acgattgcta accacctatt ggaaaaaaca ataggtcctt aaataatatt
     7621 gtcaacttca agtattgtga tgcaagcatt tagtcatgaa cgcttctcta ttctatatga
     7681 aaagccggtt ccggcctctc acctttcctt tttctcccaa tttttcagtt gaaaaaggta
     7741 tatgcgtcag gcgacctctg aaattaacaa aaaatttcca gtcatcgaat ttgattctgt
     7801 gcgatagcgc ccctgtgtgt tctcgttatg ttgaggaaaa aaataatggt tgctaagaga
     7861 ttcgaactct tgcatcttac gatacctgag tattcccaca gttaactgcg gtcaagatat
     7921 ttcttgaatc aggcgcctta gaccgctcgg ccaaacaacc aattacttgt tgagaaatag
     7981 agtataatta tcctataaat ataacgtttt tgaacacaca tgaacaagga agtacaggac
     8041 aattgatttt gaagagaatg tggattttga tgtaattgtt gggattccat ttttaataag
     8101 gcaataatat taggtatgtg gatatactag aagttctcct cgaccgtcga tagcttatcg
     8161 atgggtcctt ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat
     8221 atataaatta aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc
     8281 gaagatgtaa aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc
     8341 tgctctcagg tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa
     8401 aatcctgtga ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt
     8461 ttcttgtcta ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg
     8521 tttatttttt tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc
     8581 aaatacaaaa cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa
     8641 aagatacgag gcgcgtgtaa gttacaggca agcgatc
//
