LOCUS       V004504                 9921 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004504
VERSION     V004504
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9921)
  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 9921)
  AUTHORS   Kalivoda EJ, Horzempa J, Sadaf A, Stella NA, Nau GJ, Shanks RM.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-OCT-2010) Ophthalmology, University of Pittsburgh, 203
            Lothrop St, Pittsburgh, PA 15213, USA
REFERENCE   3  (bases 1 to 9921)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9921)
  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
            (18-OCT-2010) Ophthalmology, University of Pittsburgh, 203 Lothrop
            St, Pittsburgh, PA 15213, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9921
                     /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    1594..1610
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(2892..3593)
                     /label="dTomato"
                     /note="dimeric variant of DsRed fluorescent protein (Shaner
                     et al., 2004)"
     CDS             5380..5394
                     /label="enterokinase site"
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(5635..6327)
                     /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(6355..7350)
                     /gene="hyg"
                     /label="Hygromycin-B 7''-O-kinase"
                     /note="Hygromycin-B 7''-O-kinase from Streptomyces
                     hygroscopicus. Accession#: P09979"
     CDS             complement(7639..8730)
                     /label="LEU2"
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
     promoter        complement(8743..9147)
                     /label="LEU2 promoter"
     misc_feature    9418..9921
                     /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 tagaattctt acttgtacag ctcgtccatg ccgtacagga acaggtggtg gcggccctcg
     2941 gagcgctcgt actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccagcttg
     3001 gtgtccacgt agtagtagcc gggcagttgc acgggcttct tggccatgta gatggtcttg
     3061 aactccacca ggtagtggcc gccgtccttc agcttcaggg cctggtggat ctcgcccttc
     3121 agcacgccgt cgcgggggta caggcgctcg gtggaggcct cccagcccat ggtcttcttc
     3181 tgcattacgg ggccgtcggg ggggaagttg gtgccgcgca tcttcacctt gtagatcagc
     3241 gtgccgtcct gcagggagga gtcctgggtc acggtcacca gaccgccgtc ctcgaagttc
     3301 atcacgcgct cccacttgaa gccctcgggg aaggacagct tcttgtaatc ggggatgtcg
     3361 gcggggtgct tcacgtacgc cttggagccg tacatgaact ggggggacag gatgtcccag
     3421 gcgaagggca gggggccgcc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag
     3481 gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcatgga gccctccatg
     3541 cgcaccttga agcgcatgaa ctctttgatg acctcctcgc ccttgctcac catggtggcg
     3601 ggatcttcat atttcttcat ttgattactg atataatgat cttctttaat agcatgtagt
     3661 tgttctctta actcaggaaa ttctttgact aatggatgac gttctaacat ttaaatgctc
     3721 ctagcaattt ttgatttata acttaacttt atacatacca agagataaaa gcaaacataa
     3781 ttgtataatt aactttaatt agtgtaatta cgtatctgtg ctaaactctg ctcaaattta
     3841 atttcaccat tatcataata tgtttgtagt tgtgactcag gatgatattg atatgcctcc
     3901 taaattttta tctaaagtga atttaggagg cttacttgtc tgctttcttc attagaatca
     3961 atcctttttt aaaagtcaat attactgtaa cataaatata tattttaaaa atatcccact
     4021 ttatccaatt ttcgtttgtt gaactaatgg gtgctttagt tgaagaataa agaccacatt
     4081 aaaaaatgtg gtcttttgtg tttttttaaa ggatttgagc gtagcgaaaa atccttttct
     4141 ttcttatctt gataataagg gtaactattg ccggcgaggc tagttaccct taagttattg
     4201 gtatgactgg ttttaagcgc aaaaaaagtt gctttttcgt acctattaat gtatcgtttt
     4261 aaatgattag taaaaaacat acatagaaag gggaaaaagc aacttttttt attgtcatag
     4321 tttgtgaaaa ctaagttgtt tttatgtgtt ataacatgga aaagtatact gagaaaaaac
     4381 aaagaaatca agtatttcag aaatttatta aacgtcatat tggagagaat caaatggatt
     4441 tagttgaaga ttgcaataca tttctgtctt ttgtagctga taaaacttta gaaaaacaga
     4501 aattatataa agctaattct tgtaaaaatc gattttgtcc tgtctgtgct tggagaaaag
     4561 ctagaaaaga tgcattgggt ttatctttga tgatgcaata tattaagcag caagagaaaa
     4621 aggagtttat ctttttaact ttgactacac ctaatgtaat gagtgatgaa ttagaaaatg
     4681 aaataaaacg ttataataat tcttttagaa aacttataaa gagaaaaaaa gtaggtagtg
     4741 ttataaaggg atatgttcgt aagttagaga ttacatataa taaaaaaaga gatgattata
     4801 atcctcattt tcatgtgtta attgcagtaa ataaatcgta tttcacagat aaaagatatt
     4861 atattagcca acaagaatgg ttagatttat ggcgtgatgt aacgggcatt tcagaaataa
     4921 cacaagttca agttcaaaaa ataagacaaa ataataataa agaattatat gaaatggcta
     4981 agtattctgg taaagatagt gattatttaa taaataaatc aaaaagtctt tgatgcattt
     5041 tataaatcac ttaaaggtaa acaggtatta gtttattcag gattatttaa agaggctaaa
     5101 aagaaattaa aaaatgggga tttagattac ttaaaagaaa ttgatccaac cgtaatatat
     5161 ctatcaaatt ttttatattt ggaaacaaaa agagtattta gctagtgaac tttatgactt
     5221 aacagaacaa gaaaaaagag aaattaatca caaaatgata gacgaaatcg aggaagaaca
     5281 ataacaaaat ataagtgcta acagctgacc tcccgataac accatgtagt tattgggagg
     5341 tcagcgtgaa ttatgcacga gtattttaaa agttattgtg atgacgacga taaacgatta
     5401 tcaaaagtat aatgttaaaa tgctttatta tactaacgtt atataaacat tatactttcg
     5461 ttatacaaat tttaaccctg ttaggaacta taaaaaatca tgaaaatttt aatttgcatg
     5521 taactgggca gtgtcttaaa aaatcgacac tgaatttgct caaatttttg tttgtagaat
     5581 tagaatatat ttatttggct catatttgct ttttaaaagc acgcgtcctt attatttcag
     5641 gaaagtttcg gaggagatag tgttcggcag tttgtacatc atctgcggga tcaggtacgg
     5701 tttgatcagg ttgtagaaga tcaggtaaga catagaatcg atgtagatga tcggtttgtt
     5761 tttgttgatt tttacgtaac agttcagttg gaatttgtta cgcagaccct taaccaggta
     5821 ttctacttct tcgaaagtga aagactgggt gttcagtacg atcgatttgt tggtagagtt
     5881 tttgttgtaa tcccatttac caccatcatc catgaaccag tatgccagag acatcggggt
     5941 caggtagttt tcaaccaggt tgttcgggat ggtttttttg ttgttaacga tgaacaggtt
     6001 agccagtttg ttgaaagctt ggtgtttgaa agtctgggcg ccccaggtga ttaccaggtt
     6061 acccaggtgg ttaacacgtt gttttttgtg cggcggggac agtacccact gatcgtacag
     6121 cagacatacg tggtccatgt atgctttgtt tttccactcg aactgcatac agtaggtttt
     6181 accttcatca cgagaacgga tgtaagcatc acccaggatc agaccgatac ctgcttcgaa
     6241 ctgttcgatg ttcagttcga tcagctggga tttgtattct ttcagcagtt tagagttcgg
     6301 acccaggttc attacctggt ttttttgatg catatgcgtt ctcctacgcg ttcaggcgcc
     6361 gggggcggtg tccggcggcc cccagaggaa ctgcgccagt tcctccggat cggtgaagcc
     6421 ggagagatcc agcggggtct cctcgaacac ctcgaagtcg tgcaggaagg tgaaggcgag
     6481 cagttcgcgg gcgaagtcct cggtccgctt ccactgcgcc ccgtcgagca gcgcggccag
     6541 gatctcgcgg tcgccccgga aggcgttgag atgcagttgc accaggctgt agcgggagtc
     6601 tcccgcatag acgtcggtga agtcgacgat cccggtgacc tcggtcgcgg ccaggtccac
     6661 gaagatgttg gtcccgtgca ggtcgccgtg gacgaaccgg ggttcgcggc cggccagcag
     6721 cgtgtccacg tccggcagcc agtcctccag gcggtccagc agccggggcg agaggtagcc
     6781 ccacccgcgg tggtcctcga cggtcgccgc gcggcgttcc cgcagcagtt ccgggaagac
     6841 ctcggaatgg ggggtgagca cggtgttccc ggtcagcggc accctgtgca gccggccgag
     6901 cacccggccg agttcgcggg ccagggcgag cagcgcgttc cggtcggtcg tgccgtccat
     6961 cgcggaccgc caggtggtgc cggtcatccg gctcatcacc aggtagggcc acggccaggc
     7021 tccggtgccg ggccgcagct cgccgcggcc gaggaggcgg ggcaccggca ccggggcgtc
     7081 cgccaggacc gcgtacgcct ccgactccga cgcgaggctc tccggaccgc accagtgctc
     7141 gccgaacagc ttgatcaccg ggtcgggctc gccgaccagt acggggttgg tgctctcgcc
     7201 gggcacccgc agcaccggcg gcaccggcag cccgagctcc tccagggctc ggcgggccag
     7261 cggctcccag aattcctggt cgttccgcag gctcgcgtag gaatcatccg aatcaatacg
     7321 gtcgagaagt aacagggatt cttgtgtcat atgaatctta ctcctttgtt aaattatttt
     7381 tgtttaagca ttttgaattt gtatgcatct tagaacaatc tcaaagttcc aagctcttgc
     7441 aagtctagat attgagtcaa aaaaaaaatt ttcaactcga gcctcctttt tctccttctt
     7501 gataaatgta tgtagattgc gtatatagtt tcgtctaccc tatgaacata ttccattttg
     7561 taatttcgtg tcgtttctat tatgaatttc atttataaag tttatgtaca aatatcataa
     7621 aaaaagagaa tctttttaag caaggatttt cttaacttct tcggcgacag catcaccgac
     7681 ttcggtggta ctgttggaac cacctaaatc accagttctg atacctgcat ccaaaacctt
     7741 tttaactgca tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca atttcaacat
     7801 cattgcagca gacaagatag tggcgatagg gtcaacctta ttctttggca aatctggagc
     7861 agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa
     7921 ggacgcagat ggcaacaaac ccaaggaacc tgggataacg gaggcttcat cggagatgat
     7981 atcaccaaac atgttgctgg tgattataat accatttagg tgggttgggt tcttaactag
     8041 gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc aatgtaggga attcgttctt
     8101 gatggtttcc tccacagttt ttctccataa tcttgaagag gccaaaacat tagctttatc
     8161 caaggaccaa ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt
     8221 gattctttgc acttctggaa cggtgtattg ttcactatcc caagcgacac catcaccatc
     8281 gtcttccttt ctcttaccaa agtaaatacc tcccactaat tctctgacaa caacgaagtc
     8341 agtaccttta gcaaattgtg gcttgattgg agataagtct aaaagagagt cggatgcaaa
     8401 gttacatggt cttaagttgg cgtacaattg aagttcttta cggattttta gtaaaccttg
     8461 ttcaggtcta acactaccgg taccccattt aggaccaccc acagcaccta acaaaacggc
     8521 atcaaccttc ttggaggctt ccagcgcctc atctggaagt gggacacctg tagcatcgat
     8581 agcagcacca ccaattaaat gattttcgaa atcgaacttg acattggaac gaacatcaga
     8641 aatagcttta agaaccttaa tggcttcggc tgtgatttct tgaccaacgt ggtcacctgg
     8701 caaaacgacg atcttcttag gggcagacat aggggcagac attagaatgg tatatccttg
     8761 aaatatatat atatattgct gaaatgtaaa aggtaagaaa agttagaaag taagacgatt
     8821 gctaaccacc tattggaaaa aacaataggt ccttaaataa tattgtcaac ttcaagtatt
     8881 gtgatgcaag catttagtca tgaacgcttc tctattctat atgaaaagcc ggttccggcc
     8941 tctcaccttt cctttttctc ccaatttttc agttgaaaaa ggtatatgcg tcaggcgacc
     9001 tctgaaatta acaaaaaatt tccagtcatc gaatttgatt ctgtgcgata gcgcccctgt
     9061 gtgttctcgt tatgttgagg aaaaaaataa tggttgctaa gagattcgaa ctcttgcatc
     9121 ttacgatacc tgagtattcc cacagttaac tgcggtcaag atatttcttg aatcaggcgc
     9181 cttagaccgc tcggccaaac aaccaattac ttgttgagaa atagagtata attatcctat
     9241 aaatataacg tttttgaaca cacatgaaca aggaagtaca ggacaattga ttttgaagag
     9301 aatgtggatt ttgatgtaat tgttgggatt ccatttttaa taaggcaata atattaggta
     9361 tgtggatata ctagaagttc tcctcgaccg tcgatagctt atcgatgggt ccttttcatc
     9421 acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata
     9481 gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact
     9541 ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa
     9601 tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac
     9661 attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat
     9721 atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt
     9781 cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa
     9841 aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt
     9901 gtaagttaca ggcaagcgat c
//
