LOCUS       V004518                 8116 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004518
VERSION     V004518
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8116)
  AUTHORS   Shanks RM, Kadouri DE, MacEachran DP, O'Toole GA.
  TITLE     New yeast recombineering tools for bacteria
  JOURNAL   Plasmid 62 (2), 88-97 (2009)
   PUBMED   19477196
REFERENCE   2  (bases 1 to 8116)
  AUTHORS   Shanks RM, MacEachran DP, Powers SZ, Kadouri DE, O'Toole GA.
  TITLE     In vivo recombination vectors for modification and expression of
            genes in Gram-negative and Gram-positive bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 8116)
  AUTHORS   Shanks RM, MacEachran DP, Powers SZ, Kadouri DE, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAR-2008) Ophthalmology, University of Pittsburgh, 203
            Lothrop St., Pittsburgh, PA 15213, USA
REFERENCE   4  (bases 1 to 8116)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 8116)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid";
            date: "2009"; volume: "62"; issue: "2"; pages: "88-97"
            SGRef: number: 2; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
            (04-MAR-2008) Ophthalmology, University of Pittsburgh, 203 Lothrop
            St., Pittsburgh, PA 15213, USA"
            SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(238..1656)
                     /label="SacB"
                     /note="secreted levansucrase that renders bacterial growth
                     sensitive to sucrose"
     promoter        complement(1657..2102)
                     /label="sacB promoter"
                     /note="sacB promoter and control region"
     oriT            complement(2476..2585)
                     /direction=LEFT
                     /label="oriT"
                     /note="incP origin of transfer"
     terminator      complement(3006..3033)
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(3125..3211)
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     3561..3577
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     misc_feature    complement(3581..3637)
                     /label="MCS"
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3647..3663)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(3671..3687)
                     /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(3695..3725)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3740..3761)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        complement(3812..3830)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      4038..4426
                     /label="R6K gamma ori"
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     CDS             4777..5568
                     /label="NeoR/KanR"
                     /note="aminoglycoside phosphotransferase"
     CDS             5868..6239
                     /gene="rpsL"
                     /label="Small ribosomal subunit protein uS12"
                     /note="Small ribosomal subunit protein uS12 from Salmonella
                     paratyphi A (strain ATCC 9150 / SARB42). Accession#:
                     Q5PIW1"
     CDS             complement(6505..7305)
                     /label="URA3"
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(7306..7526)
                     /label="URA3 promoter"
     misc_feature    7573..8076
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
       61 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
      121 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
      181 atcagagcag attgtactga gagtgcacca taatcggcat tttcttttgc gtttttattt
      241 gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc
      301 tttgatgttc agcaggaagc taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct
      361 gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg
      421 tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt
      481 gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc
      541 gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acagatacca
      601 tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc
      661 aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag
      721 agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc
      781 ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc
      841 ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc
      901 ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc
      961 gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc
     1021 ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt
     1081 cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc
     1141 gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc
     1201 atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat
     1261 agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg catttttagg
     1321 atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag tgccgtcagc
     1381 gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga tatttttaat
     1441 tgtggacgaa tcgaactcag gaacttgata tttttcattt ttttgctgtt cagggatttg
     1501 cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg gcttttggtt
     1561 cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag taaaggttaa
     1621 tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct tttttatgta
     1681 ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt tagttacgca
     1741 caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact ttgcccttta
     1801 cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac ttttcgacct
     1861 cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt gtttgataga
     1921 aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta tctgtttctt
     1981 ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc tttttaatca
     2041 caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg caggtatatg
     2101 tgatgggtta aaaaggatcg atcctctagc tagagtcgat cttcgccagc agggcgagga
     2161 tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt gagccagagt ttcagcaggc
     2221 cgcccaggcg gcccaggtcg ccattgatgc gggccagctc gcggacgtgc tcatagtcca
     2281 cgacgcccgt gattttgtag ccctggccga cggccagcag gtaggccgac aggctcatgc
     2341 cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc tggaaggcag tacaccttga
     2401 taggtgggct gcccttcctg gttggcttgg tttcatcagc catccgcttg ccctcatctg
     2461 ttacgccggc ggtagccggc cagcctcgca gagcaggatt cccgttgagc accgccaggt
     2521 gcgaataagg gacagtgaag aaggaacacc cgctcgcggg tgggcctact tcacctatcc
     2581 tgcccggctg acgccgttgg atacaccaag gaaagtctac acgaaccctt tggcaaaatc
     2641 ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg accccgaagc
     2701 agggttatgc agcggaaaag cgctgcttcc ctgctgtttt gtggaatatc taccgactgg
     2761 aaacaggcaa atgcaggaaa ttactgaact gaggggacag gcgagagacg atgccaaaga
     2821 gctacaccga cgagctggcc gagtgggttg aatcccgcgc ggccaagaag cgccggcgtg
     2881 atgaggctgc ggttgcgttc ctggcggtga gggcggatgt cgatatgcgt aaggagaaaa
     2941 taccgcatca ggcgcatatt tgaatgtatt tagaaaaata aacaaaaaga gtttgtagaa
     3001 acgcaaaaag gccatccgtc aggatggcct tctgcttaat ttgatgcctg gcagtttatg
     3061 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg
     3121 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     3181 agtctttcga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg
     3241 ggagacccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca
     3301 ggtgggacca ccgcgctact gccgccaggc aaattctgtt ttatcagacc gcttctgcgt
     3361 tctgatttaa tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca gccaagctcg
     3421 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3481 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3541 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagcttgcat gcctgcaggt
     3601 cgactctaga ggatccccgg gtaccgagct cgaattcgta atcatggtca tagctgtttc
     3661 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3721 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3781 ccgctttcca gtcgggaaac cgctctagac ccctatagtg agtcgtatta caaactggaa
     3841 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3901 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3961 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaacca gatccccacg
     4021 atgcgtccgg cgtagaggat ctgaagatca gcagttcaac ctgttgatag tacgtactaa
     4081 gctctcatgt ttcacgtact aagctctcat gtttaacgta ctaagctctc atgtttaacg
     4141 aactaaaccc tcatggctaa cgtactaagc tctcatggct aacgtactaa gctctcatgt
     4201 ttcacgtact aagctctcat gtttgaacaa taaaattaat ataaatcagc aacttaaata
     4261 gcctctaagg ttttaagttt tataagaaaa aaaagaatat ataaggcttt taaagctttt
     4321 aaggtttaac ggttgtggac aacaagccag ggatgtaacg cactgagaag cccttagagc
     4381 ctctcaaagc aattttcagt gacacaggaa cacttaacgg ctgacagacg ctgccgcaag
     4441 cactcagggc gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa
     4501 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     4561 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     4621 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     4681 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     4741 agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4801 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4861 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4921 gtcaagaccg acctgtccgg tgccctgaat gaactccaag acgaggcagc gcggctatcg
     4981 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     5041 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     5101 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     5161 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     5221 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5281 gaactgttcg ccaggctcaa ggcgcggatg cccgacggcg aggatctcgt cgtgacccat
     5341 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5401 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5461 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5521 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5581 tggggtacgc gtctaggcgg ccgcgaagtt cctattccga agttcctcta gaagcttctg
     5641 cagacgggtt tgactggtca aatttcgaac gacatcattc tcattgacta tactgatttc
     5701 gtcagactta cggttaagca ccccagccag atggcctggt gatggcggga tcgttgtata
     5761 tttcttgaca ccttttcggc atcgccctaa aattcggcgt cctcatattg tgtgaggacg
     5821 ttttattacg tgtttacgaa gcaaaagcta aaaccaggag ctatttaatg gcaacagtta
     5881 accagctggt acgcaaacca cgtgctcgca aagttgcgaa aagcaacgtg cctgcgctgg
     5941 aagcatgccc gcaaaaacgt ggcgtatgta ctcgtgtata tactaccact cctaaaaaac
     6001 cgaactccgc gctgcgtaaa gtatgccgtg ttcgtctgac taacggtttc gaagtgactt
     6061 cctacatcgg tggtgaaggt cacaacctgc aggagcactc cgtgatcctg atccgtggcg
     6121 gtcgtgttaa agacctcccg ggtgttcgtt accacaccgt acgtggtgcg cttgactgct
     6181 ccggcgttaa agaccgtaag caggctcgtt ccaagtatgg cgtgaagcgt cctaaggctt
     6241 aatggttctc cgttaagtaa ggccaaacgt tttaacttaa atgtcaaact aaactcgtag
     6301 agttttggac aatcctgatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6361 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6421 agtgggtaat aactgatata attaaattga agctctaatt tgtgagttta gtatacatgc
     6481 atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat atgcttccca
     6541 gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg caaatagtcc
     6601 tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg ttctatactg
     6661 ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc
     6721 gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa caaaatcttt
     6781 gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata gggagccctt
     6841 gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt cttctgccgc
     6901 ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg taatgtctgc
     6961 ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat taccaatgtc
     7021 agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg cctttagcgg
     7081 cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt ttagtaaaca
     7141 aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac gaacatccaa
     7201 tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg cagcaacagg
     7261 actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt atcttcgttt
     7321 cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc atgtttcttc
     7381 aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc gttcttcctt
     7441 ctgttcggag attaccgaat caaaaaaatt tcaaagaaac cgaaatcaaa aaaaagaata
     7501 aaaaaaaaat gatgaattga attgaaaagc tagcttatcg atgggtcctt ttcatcacgt
     7561 gcggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt aatataaata
     7621 tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt ttgttttccg
     7681 aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct tgctcttcct
     7741 gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc acacacgaaa
     7801 atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt aatctgcttt
     7861 tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt aaacctttgt
     7921 ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt ctaaaatcca
     7981 aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc catcattaaa
     8041 agatacgagg cgcgtgtaag ttacaggcaa gcgatctcta agaaaccatt attatcatga
     8101 cattaaccta taaaaa
//
