LOCUS       V004515                 8636 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004515
VERSION     V004515
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8636)
  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 8636)
  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  JOURNAL   Unpublished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 8636)
  AUTHORS   Shanks RM, MacEachran DP, Powers SZ, Kadouri DE, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-MAR-2008) Ophthalmology, University of Pittsburgh, 203
            Lothrop St., Pittsburgh, PA 15213, USA
REFERENCE   4  (bases 1 to 8636)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 8636)
  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            "Unpublished"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
            (03-MAR-2008) Ophthalmology, University of Pittsburgh, 203 Lothrop
            St., Pittsburgh, PA 15213, USA"
            SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        196..416
                     /label="URA3 promoter"
     CDS             417..1217
                     /label="URA3"
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     1951..1967
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        1977..1995
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2021..2037
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     rep_origin      2274..2431
                     /label="palA lagging strand origin"
                     /note="palA lagging strand origin"
     CDS             complement(2573..3220)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     CDS             complement(3389..3772)
                     /codon_start=1
                     /gene="rep"
                     /product="replication protein"
                     /label="rep"
                     /protein_id="ACB45180.1"
                     /translation="MHLILSNMTFNKFLKYLISLFFLSILFHVITHKNNLVFTNYDNKK
                     SCFFPFLCMFFTSHLKRYINRYEKATFFALKTSHTNNLRVTSLAGNSYPYYQDKKEKDF
                     SLRSNPLKKHKRPHFLMWSLFFN"
     gene            complement(3389..3772)
                     /gene="rep"
                     /label="rep"
     rep_origin      3427..3555
                     /label="oriV for pC194"
                     /note="oriV for pC194"
     CDS             3677..4366
                     /codon_start=1
                     /gene="pre"
                     /product="Pre"
                     /label="pre"
                     /note="pC194 OrfA"
                     /protein_id="ACB45181.1"
                     /translation="MCYNMEKYTEKKQRNQVFQKFIKRHIGENQMDLVEDCNTFLSFVA
                     DKTLEKQKLYKANSCKNRFCPVCAWRKARKDALGLSLMMQYIKQQEKKEFIFLTLTTPN
                     VMSDELENEIKRYNNSFRKLIKRKKVGSVIKGYVRKLEITYNKKRDDYNPHFHVLIAVN
                     KSYFTDKRYYISQQEWLDLWRDVTGISEITQVQVQKIRQNNNKELYEMAKYSGKDSDYL
                     INKSKSL"
     gene            3677..4366
                     /gene="pre"
                     /label="pre"
     misc_feature    4613..4699
                     /label="palB"
                     /note="palB"
     CDS             4713..4727
                     /label="enterokinase site"
                     /note="enterokinase recognition and cleavage site"
     primer_bind     complement(4981..4997)
                     /label="SK primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        complement(5034..5052)
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5073..5089)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(5097..5113)
                     /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(5121..5151)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5166..5187)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5475..6063)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(6237..7094)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(7095..7199)
                     /label="AmpR promoter"
     rep_origin      complement(7226..8568)
                     /direction=LEFT
                     /label="2u ori"
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata
     2041 agcttctgta ggtttttagg cataaaacta tatgatttac ccctaaatct ttaaaatgcc
     2101 ccttaaaatt caaaataaag gcatttaaaa tttaaatatt tcttgtgata aagtttgtta
     2161 aaaaggagtg gttttatgac tgttatgtgg ttatcgatta taggtatgtg gttttgtatt
     2221 ggaatggcat tttttgctat caaggttatt aaaaataaaa attagaccac gcatttatgc
     2281 cgagaaaatt tattgtgcgt tgagaagaac ccttaactaa acttgcagac gaatgtcggc
     2341 atagcgtgag ctattaagcc gaccattcga caagttttgg gattgttaag ggttccgagg
     2401 ctcaacgtca ataaagcaat tggaataaag aagcgaaaaa ggagaagtcg gttcagaaaa
     2461 agaaggatat ggatctggag ctgtaatata aaaaccttct tcaactaacg gggcaggtta
     2521 gtgacattag aaaaccgact gtaaaaagta cagtcggcat tatctcatat tataaaagcc
     2581 agtcattagg cctatctgac aattcctgaa tagagttcat aaacaatcct gcatgataac
     2641 catcacaaac agaatgatgt acctgtaaag atagcggtaa atatattgaa ttacctttat
     2701 taatgaattt tcctgctgta ataatgggta gaaggtaatt actattatta ttgatattta
     2761 agttaaaccc agtaaatgaa gtccatggaa taatagaaag agaaaaagca ttttcaggta
     2821 taggtgtttt gggaaacaat ttccccgaac cattatattt ctctacatca gaaaggtata
     2881 aatcataaaa ctctttgaag tcattcttta caggagtcca aataccagag aatgttttag
     2941 atacaccatc aaaaattgta taaagtggct ctaacttatc ccaataacct aactctccgt
     3001 cgctattgta accagttcta aaagctgtat ttgagtttat cacccttgtc actaagaaaa
     3061 taaatgcagg gtaaaattta tatccttctt gttttatgtt tcggtataaa acactaatat
     3121 caatttctgt ggttatacta aaagtcgttt gttggttcaa ataatgatta aatatctctt
     3181 ttctcttcca attgtctaaa tcaattttat taaagttcat ttgatatgcc tcctaaattt
     3241 ttatctaaag tgaatttagg aggcttactt gtctgctttc ttcattagaa tcaatccttt
     3301 tttaaaagtc aatattactg taacataaat atatatttta aaaatatccc actttatcca
     3361 attttcgttt gttgaactaa tgggtgcttt agttgaagaa taaagaccac attaaaaaat
     3421 gtggtctttt gtgttttttt aaaggatttg agcgtagcga aaaatccttt tctttcttat
     3481 cttgataata agggtaacta ttgccggcga ggctagttac ccttaagtta ttggtatgac
     3541 tggttttaag cgcaaaaaaa gttgcttttt cgtacctatt aatgtatcgt tttaaatgac
     3601 tagtaaaaaa catacataga aaggggaaaa agcaactttt tttattgtca tagtttgtga
     3661 aaactaagtt gtttttatgt gttataacat ggaaaagtat actgagaaaa aacaaagaaa
     3721 tcaagtattt cagaaattta ttaaacgtca tattggagag aatcaaatgg atttagttga
     3781 agattgcaat acatttctgt cttttgtagc tgataaaact ttagaaaaac agaaattata
     3841 taaagctaat tcttgtaaaa atcgattttg tcctgtctgt gcttggagaa aagctagaaa
     3901 agatgcattg ggtttatctt tgatgatgca atatattaag cagcaagaga aaaaggagtt
     3961 tatcttttta actttgacta cacctaatgt aatgagtgat gaattagaaa atgaaataaa
     4021 acgttataat aattctttta gaaaacttat aaagagaaaa aaagtaggta gtgttataaa
     4081 gggatatgtt cgtaagttag agattacata taataaaaaa agagatgatt ataatcctca
     4141 ttttcatgtg ttaattgcag taaataaatc gtatttcaca gataaaagat attatattag
     4201 ccaacaagaa tggttagatt tatggcgtga tgtaacgggc atttcagaaa taacacaagt
     4261 tcaagttcaa aaaataagac aaaataataa taaagaatta tatgaaatgg ctaagtattc
     4321 tggtaaagat agtgattatt taataaataa atcaaaaagt ctttgatgca ttttataaat
     4381 cacttaaagg taaacaggta ttagtttatt caggattatt taaagaggct aaaaagaaat
     4441 taaaaaatgg ggatttagat tacttaaaag aaattgatcc aaccgtaata tatctatcaa
     4501 attttttata tttggaaaca aaaagagtat ttagctagtg aactttatga cttaacagaa
     4561 caagaaaaaa gagaaattaa tcacaaaatg atagacgaaa tcgaggaaga acaataacaa
     4621 aatataagtg ctaacagctg acctcccgat aacaccatgt agttattggg aggtcagcgt
     4681 gaattatgca cgagtatttt aaaagttatt gtgatgacga cgataaacga ttatcaaaag
     4741 tataatgtta aaatgcttta ttatactaac gttatataaa cattatactt tcgttataca
     4801 aattttaacc ctgttaggaa ctataaaaaa tcatgaaaat tttaatttgc atgtaactgg
     4861 gcagtgtctt aaaaaatcga cactgaattt gctcaaattt ttgtttgtag aattagaata
     4921 tatttatttg gctcatattt gctttttaaa agcttgatat cgaattcctg cagcccgggg
     4981 gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag
     5041 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5101 tatccgctca caattccaca caacatagga gccggaagca taaagtgtaa agcctggggt
     5161 gcctaatgag tgaggtaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5221 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5281 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5341 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5401 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5641 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5881 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6121 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6181 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6361 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6541 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7081 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7201 acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg tagaacaaaa
     7261 atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca
     7321 gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa
     7381 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca
     7441 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg
     7501 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt
     7561 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt
     7621 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc
     7681 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc
     7741 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata
     7801 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat
     7861 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt
     7921 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag
     7981 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat
     8041 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc
     8101 aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca
     8161 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact
     8221 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc
     8281 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata
     8341 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct
     8401 atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg
     8461 gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat
     8521 tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat actaagaaac
     8581 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
