LOCUS       sequence-73679-e        6861 bp    DNA     circular SYN 14-APR-2025
DEFINITION  overespression of Flp step-arrest mutant in yeast, URA selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6861)
  AUTHORS   Tsalik EL, Gartenberg MR
  TITLE     Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted 
            DNA damage.
  JOURNAL   Yeast. 1998 Jun 30;14(9):847-52.
  PUBMED    9818722
REFERENCE   2  (bases 1 to 6861)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Yeast."; 
            date: "1998-06-30"; volume: "14(9)"; pages: "847-52"
COMMENT     Sequence Label: pBIS-GALkFLP(URA)
FEATURES             Location/Qualifiers
     source          1..6861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1977..1996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1977..1995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2020..2036
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2021..2037
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2250..2580
                     /gene="S. cerevisiae GAL10"
                     /label=GAL10 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2250..2367
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             2588..3859
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSLIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(4087..4107)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4087..4105)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4126..4142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4126..4142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4139..4161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4150..4166
                     /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(join(4174..4180,4181..4198,4199..4204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4357..4374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4528..5116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4608..4627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5287..6078,6079..6147))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5910..5929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6148..6252)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6289..6792
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6834..6852
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
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 agcttgatat cgaattcctg cagcccgggg gatccaaaaa tcatcgcttc gctgattaat
     2101 taccccagaa ataaggctaa aaaactaatc gcattatcat cctatggttg ttaatttgat
     2161 tcgttcattt gaaggtttgt ggggccaggt tactgccaat ttttcctctt cataaccata
     2221 aaagctagta ttgtagaatc tttattgttc ggagcagtgc ggcgcgaggc acatctgcgt
     2281 ttcaggaacg cgaccggtga agacgaggac gcacggagga gagtcttcct tcggagggct
     2341 gtcacccgct cggcggcttc taatccgtac ttcaatatag caatgagcag ttaagcgtat
     2401 tactgaaagt tccaaagaga aggttttttt aggctaagat aatggggctc tttacatttc
     2461 cacaacatat aagtaagatt agatatggat atgtatatgg atatgtatat ggtggtaatg
     2521 ccatgtaata tgattattaa acttctttgc gtccatccaa aaaaaaagta agaatttttg
     2581 ggtcgacatg ccacaatttg gtatattatg taaaacacca cctaaggtgc ttgttcgtca
     2641 gtttgtggaa aggtttgaaa gaccttcagg tgagaaaata gcattatgtg ctgctgaact
     2701 aacctattta tgttggatga ttacacataa cggaacagca atcaagagag ccacattcat
     2761 gagctataat actatcataa gcaattcgct gagtttcgat attgtcaata aatcactcca
     2821 gtttaaatac aagacgcaaa aagcaacaat tctggaagcc tcattaaaga aattgattcc
     2881 tgcttgggaa tttacaatta ttccttacta tggacaaaaa catcaatctg atatcactga
     2941 tattgtaagt agtttgcaat tacagttcga atcatcggaa gaagcagata agggaaatag
     3001 ccacagtaaa aaaatgctta aagcacttct aagtgagggt gaaagcatct gggagatcac
     3061 tgagaaaata ctaaattcgt ttgagtatac ttcgagattt acaaaaacaa aaactttata
     3121 ccaattcctc ttcctagcta ctttcatcaa ttgtggaaga ttcagcgata ttaagaacgt
     3181 tgatccgaaa tcatttaaat tagtccaaaa taagtatctg ggagtaataa tccagtgttt
     3241 agtgacagag acaaagacaa gcgttagtag gcacatatac ttctttagcg caaggggtag
     3301 gatcgatcca cttgtatatt tggatgaatt tttgaggaat tctgaaccag tcctaaaacg
     3361 agtaaatagg accggcaatt cttcaagcaa taaacaggaa taccaattat taaaagataa
     3421 cttagtcaga tcgtacaata aagctttgaa gaaaaatgcg ccttattcaa tctttgctat
     3481 aaaaaatggc ccaaaatctc tcattggaag acatttgatg acctcatttc tttcaatgaa
     3541 gggcctaacg gagttgacta atgttgtggg aaattggagc gataagcgtg cttctgccgt
     3601 ggccaggaca acgtatactc atcagataac agcaatacct gatcactact tcgcactagt
     3661 ttctcggtac tatgcatatg atccaatatc aaaggaaatg atagcattga aggatgagac
     3721 taatccaatt gaggagtggc agcatataga acagctaaag ggtagtgctg aaggaagcat
     3781 acgatacccc gcatggaatg ggataatatc acaggaggta ctagactacc tttcatccta
     3841 cataaataga cgcatataag tacgcattta agcataaaca cgcactatgc cgttcttctc
     3901 atgtatatat atatacaggc aacacgcaga tataggtgcg acgtgaacag tgagctgtat
     3961 gtgcgcagct cgcgttgcat tttcggaagc gctcgttttc ggaaacgctt tgaagttcct
     4021 attccgaagt tcctattctc tagttctaga gcggccgcca ccgcggtgga gctccagctt
     4081 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4141 tgtgtgaaat tgttatccgc tcacaattcc acacaacata ggagccggaa gcataaagtg
     4201 taaagcctgg ggtgcctaat gagtgaggta actcacatta attgcgttgc gctcactgcc
     4261 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4321 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4381 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4441 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4501 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4561 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4621 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4681 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4741 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4801 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4861 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4921 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4981 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5041 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5101 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5161 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5221 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5281 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5341 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5401 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5461 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5521 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5581 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5641 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5701 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5761 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5821 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5881 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5941 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6001 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6061 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6121 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6181 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6241 aggggttccg cgcacatttc cccgaaaagt gccacctggg tccttttcat cacgtgctat
     6301 aaaaataatt ataatttaaa ttttttaata taaatatata aattaaaaat agaaagtaaa
     6361 aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac tctaggggga
     6421 tcgccaacaa atactacctt ttatcttgct cttcctgctc tcaggtatta atgccgaatt
     6481 gtttcatctt gtctgtgtag aagaccacac acgaaaatcc tgtgatttta cattttactt
     6541 atcgttaatc gaatgtatat ctatttaatc tgcttttctt gtctaataaa tatatatgta
     6601 aagtacgctt tttgttgaaa ttttttaaac ctttgtttat ttttttttct tcattccgta
     6661 actcttctac cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa
     6721 acacagagta aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac
     6781 aggcaagcga tccgtcctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     6841 gtatcacgag gccctttcgt c
//
