LOCUS       sequence-441038-        6598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for selection/counterslection in budding yeast, containing 
            URA3 and LEU2 markers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  AUTHORS   Stojkovic L, Gligorovski V, Geramimanesh M, Labagnara M, Rahi SJ
  TITLE     Automated plasmid design for marker-free genome editing in budding 
            yeast.
  JOURNAL   G3 (Bethesda). 2024 Dec 17:jkae297. doi: 10.1093/g3journal/jkae297.
  PUBMED    39688855
REFERENCE   2  (bases 1 to 6598)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "G3 
            (Bethesda). 2024 Dec 17:jkae297. doi: 10.1093/g3journal/jkae297."
COMMENT     Sequence Label: pETURALEU
FEATURES             Location/Qualifiers
     source          1..6598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..108
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     17..33
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     18..34
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(68..84)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(68..84)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(121..141)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(121..139)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(160..176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(160..176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(173..195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    184..200
                     /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(208..214,215..232,233..238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    253..274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(391..408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(562..1150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(642..661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1321..2112,2113..2181))
                     /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     1944..1963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2182..2286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2354..2372
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2410..2432)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2483..3286)
                     /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"
     promoter        complement(3287..3502)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     3634..3653
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4147..5241)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5242..5649)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(5950..6405)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6087..6108)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6299..6318
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(6382..6598,1..164))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     6531..6553
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6545..6562
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6546..6562
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6572..6591
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6572..6590
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga attcctgcag
       61 cccgggggat ccactagttc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
      121 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
      181 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
      241 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
      301 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
      361 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
      421 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      481 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      541 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      601 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      661 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      721 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
      781 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
      841 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
      901 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
      961 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1021 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1081 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1141 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1201 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1261 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1321 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1381 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1441 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     1501 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1561 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1621 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1681 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     1741 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1801 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1861 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1921 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1981 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2041 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2101 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2161 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2221 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2281 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     2341 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     2401 cggtgaaaac ctctgacaca tgcagctccc ggactctaat ttgtgagttt agtatacatg
     2461 catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa tatgcttccc
     2521 agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt gcaaatagtc
     2581 ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg gttctatact
     2641 gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata atcaaccaat
     2701 cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata acaaaatctt
     2761 tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat agggagccct
     2821 tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact tcttctgccg
     2881 cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc gtaatgtctg
     2941 cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta ttaccaatgt
     3001 cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat gcctttagcg
     3061 gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac
     3121 aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta cgaacatcca
     3181 atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg gcagcaacag
     3241 gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt tatcttcgtt
     3301 tcctgcaggt ttttgttctg tgcagttggg ttaagaatac tgggcaattt catgtttctt
     3361 caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt cgttcttcct
     3421 tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa aaaaaagaat
     3481 aaaaaaaaaa tgatgaattg aaaaggtggt atggtgcact ctcagtactc cgggagacgg
     3541 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     3601 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     3661 tgcaccatat cgactacgtc gttaaggccg tttctgacag agtaaaattc ttgagggaac
     3721 tttcaccatt atgggaaatg gttcaagaag gtattgactt aaactccatc aaatggtcag
     3781 gtcattgagt gttttttatt tgttgtattt tttttttttt agagaaaatc ctccaatata
     3841 taaattagga atcatagttt catgattttc tgttacacct aactttttgt gtggtgccct
     3901 cctccttgtc aatattaatg ttaaagtgca attctttttc cttatcacgt tgagccatta
     3961 gtatcaattt gcttacctgt attcctttac atcctccttt ttctccttct tgataaatgt
     4021 atgtagattg cgtatatagt ttcgtctacc ctatgaacat attccatttt gtaatttcgt
     4081 gtcgtttcta ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga
     4141 atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt
     4201 actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc
     4261 atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc
     4321 agacaagata gtggcgatag ggttgacctt attctttggc aaatctggag cagaaccgtg
     4381 gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga
     4441 tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa
     4501 catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc
     4561 ggcagaatca atcaattgat gttgaacctt caatgtaggg aattcgttct tgatggtttc
     4621 ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca
     4681 aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg
     4741 cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt
     4801 tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt
     4861 agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg
     4921 tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct
     4981 aacactaccg gtaccccatt taggaccacc cacagcacct aacaaaacgg catcagcctt
     5041 cttggaggct tccagcgcct catctggaag tggaacacct gtagcatcga tagcagcacc
     5101 accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt
     5161 aagaacctta atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac
     5221 gatcttctta ggggcagaca ttagaatggt atatccttga aatatatata tatatattgc
     5281 tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa
     5341 aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc
     5401 atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct ttcctttttc
     5461 tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa
     5521 tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc gttatgttga
     5581 ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt
     5641 cccacagtta actgcggtca agatatttct tgaatcaggc gccttagacc gctcggccaa
     5701 acaaccaatt acttgttgag aaatagagta taattatcct ataaatataa cgtttttgaa
     5761 cacacatgaa caaggaagta caggacaatt gattttgaag agaatgtgga ttttgatgta
     5821 attgttggga ttccattttt aataaggcaa taatattagg tatgtagata tactagaagt
     5881 tctcctcgac cggtcgatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     5941 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6001 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6061 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6121 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6181 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6241 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6301 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6361 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     6421 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     6481 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     6541 acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattg
//
