LOCUS       sequence-363407-        9748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing a biosensor for acetic acid: 
            BM3R1-Haa1-mTurquoise2 under RET2p and mCherry under HREsYGP1p with 
            flanks for genome integration into HO locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9748)
  AUTHORS   Mormino M, Lenitz I, Siewers V, Nygard Y
  TITLE     Identification of acetic acid sensitive strains through 
            biosensor-based screening of a Saccharomyces cerevisiae CRISPRi 
            library.
  JOURNAL   Microb Cell Fact. 2022 Oct 15;21(1):214. doi: 
            10.1186/s12934-022-01938-7.
  PUBMED    36243715
REFERENCE   2  (bases 1 to 9748)
  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: "Microb Cell
            Fact."; date: "2022-10-15"; volume: "21(1):214. doi"; pages: " 
            10.1186/s12934-022-01938-7"
COMMENT     Sequence Label: pMM4_14
FEATURES             Location/Qualifiers
     source          1..9748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4835..5545
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVDGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4980..4998)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5248..5267
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6009..6812
                     /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"
     CDS             complement(7454..8269)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7454..8269)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     8177..8196
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8261..8280
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8494..9082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8983..9002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgcagacga acgacgatgg cttcttatct cacttcaata gtactttcca ccggttatac
       61 ttccggcttt tccctattaa tacaagctac aatttcaatg ggtggcaaat aatgtgtaga
      121 atagaaaata agccgacagg gtaataaaga aaatttttag aaaaaaaagg ttagatggct
      181 tatttaagtt acaggctagc gaaaaaagga acttcagggc aagtaaagtg tttgattggg
      241 cactagcatg gcttataaag gcgagcaatt gtcgaaacta attaatgttg tacggactat
      301 tgctgtcatc tcgtggtaaa tgcgtgttcc aggtcgaata ctacttgcac acaggcgagc
      361 ggggccccat aaaagtgttg ccgatttgtt aagttgtctt ttcggttttt ctactctgtt
      421 attccttact tcccttttta agaactcttt ttatccttca tttaggatct tgcacgtttc
      481 cgcctcatca cttgaattaa aacatgtctc tgtcagtaaa ccttggcgtt tctattgttc
      541 ttcatagttc aacttttatt attacccgcc ctgcgcgttt acatttttcc agcaacagcc
      601 agcgaaaaat tagaaaatct ggttgttgac acctcaagaa caagggcaat tagcctcagc
      661 gtcgaatata gatcatatta gaatacctat agctccatca aaagaaatac acaagatcta
      721 tggaatctac tcctactaag caaaaagcca tcttctctgc ctccttgttg ttgttcgctg
      781 agagaggttt cgacgctacc actatgccaa tgattgccga aaacgctaag gttggtgctg
      841 gtactatcta cagatacttc aagaacaaag aatccttagt taatgagttg tttcaacaac
      901 acgtcaatga atttttgcaa tgtatcgaat ctggtttggc taacgaaaga gatggttaca
      961 gggacggttt ccaccacatc ttcgaaggta tggtcacctt caccaagaac catcctagag
     1021 ccttaggttt catcaagacc cactctcaag gtactttttt gaccgaagaa tccagattgg
     1081 cttatcaaaa gttggtcgaa tttgtctgta ctttcttcag agaaggtcaa aagcaaggtg
     1141 tcatcagaaa tttgccagaa aacgctttga ttgctatctt gttcggttct ttcatggaag
     1201 tctacgaaat gattgaaaac gattatttgt ctttgactga tgaattatta accggtgttg
     1261 aggaatcctt gtgggctgcc ttgtctcgtc aatctggttc tatggtcttg ataaatggca
     1321 taaagtatgc ctgtgagagg tgcataagag gccatagagt aacaacatgc aatcatacag
     1381 atcaaccgct tatgatgatc aaacccaaag gtagaccttc cactacatgc gactattgta
     1441 aacaacttcg aaaaaacaag aatgcaaatc ctgaaggtgt ttgcacgtgt ggccggctag
     1501 agaagaaaaa actggcacag aaagccaaag aagaagcaag agctaaagcc aaagaaaaac
     1561 aaagaaaaca gtgtacctgc gggactgatg aggtttgcaa atatcatgct caaaagagac
     1621 atctaagaaa gtccccttca agttctcaaa agaaaggaag atccatttct cgttctcaac
     1681 caatgtttga aagggtattg tcttctactt cacttgacag caatatgtta tccggccacg
     1741 gagcactatc agatacctct agcatactga cgagcacatt tttagacagt gagccgggtg
     1801 ttggtaaaat ttcaaaagat taccatcatg tcccttcatt ggcctccatt tcatccttac
     1861 aatcctcgca atcgttagat caaaatttca gtataccaca aagcccgccg ttatcttcaa
     1921 tgtcatttaa ttttctcacg ggaaatatca atgaaaccaa ccaaaatcac agtaatcatc
     1981 agcattcaaa atcaggcaat aactggcaag atagttcggt aagcttgcca gcgaaagctg
     2041 attcacgtct taacatgatg gataaaaaca actctgtggg tcttgaccta ttaggccatt
     2101 caaaacgaat atcgccgata tcaaactctc gtgtgggcga agttagcgtt ccgctagaag
     2161 aatatattcc ttctgacatt gatggggttg gaagagttac tgataaaagc tctttggtct
     2221 acgattggcc atttgatgaa agtattgaga gaaatttcag tacaaccgca accgctgcaa
     2281 ctggtgaaag taagttcgac attaacgaca actgtaatag aattaatagc aaaagttata
     2341 gtaagactaa tagtatgaat ggaaacggta tgaacaatag caataataat aatatcaaca
     2401 gtaatggcaa cgacaagaac aataacaact cttctagaca agaacatcaa ggaaatggac
     2461 tatttgacat gtttacagat tcatcgtcga tttcaacgct ttcccgtgca aacttattat
     2521 tgcaagaaaa aattggttcg caagaaaact ctgtcaaaca agaaaactat tcgaaaaatc
     2581 ctcaacttcg tcatcaatta acttccagaa gtagatcatt tattcatcat ccggcaaacg
     2641 agtatttgaa gaatactttt ggaaattcac atagtaatga catcggaaag ggagttgaag
     2701 tgctatcttt gacaccgagt tttatggata ttcccgaaaa agaaagagaa acggaaagat
     2761 cgccatcatc caattacatt actgacagac ctttcactcg aaaacctaga tcttctagca
     2821 ttgacgtaaa ccataggtat ccacctatgg caccaacaac cgtagcgaca tctcccggtg
     2881 cattgaacaa tgccgtagca agcaatctcg acgatcaact gagtttaaca tcactaaact
     2941 ctcagccatc atcgatagca aatatgatga tggacccttc aaacctagct gagcaaagtt
     3001 ctattcattc agttcctcag tcaataaact ctccgagaat gcctaaaact ggaagtcgcc
     3061 aagacaagaa cattcacact aagaaggaag aaagaaatcc gctaaataac atacacgatc
     3121 tgtcacaatt ggaaaatgta ccagacgaga tgaaccaaat gttctcccca ccattaaaaa
     3181 gtatgaatag accggatgcc ataagggaaa attcatctag tagtaatttc ataatccaag
     3241 gaaatagcat gatctctacg ccttccggaa ggaatgacct tccagatacc tctccaatga
     3301 gtagtattca aacagcgtca ccaccaagtc aattactgac cgatcaagga tttgcggatt
     3361 tggataattt catgtcttcg ttaggttctg tttctaaagg tgaagaatta ttcactggtg
     3421 ttgtcccaat tttggttgaa ttagatggtg atgttaatgg tcacaaattt tctgtctccg
     3481 gtgaaggtga aggtgatgct acttacggta aattgacctt aaaatttatt tgtactactg
     3541 gtaaattgcc agttccatgg ccaaccttag tcactacttt atcttggggt gttcaatgtt
     3601 ttgcaagata cccagatcat atgaaacaac atgacttttt caagtctgcc atgccagaag
     3661 gttatgttca agaaagaact atttttttca aagatgacgg taactacaag accagagctg
     3721 aagtcaagtt tgaaggtgat accttagtta atagaatcga attaaaaggt attgatttta
     3781 aagaagatgg taacatttta ggtcacaaat tggaatacaa ttatttctct gacaatgttt
     3841 acatcactgc tgacaaacaa aagaatggta tcaaagctaa cttcaaaatt agacacaaca
     3901 ttgaagatgg tggtgttcaa ttagctgacc attatcaaca aaatactcca attggtgatg
     3961 gtccagtctt gttaccagac aaccattact tatccactca atctaagtta tccaaagatc
     4021 caaacgaaaa gagggaccac atggtcttgt tagaatttgt tactgctgct ggtattacct
     4081 tgggtatgga tgaattgtac aaaggatcct aactcgagag cttttgatta agccttctag
     4141 tccaaaaaac acgttttttt gtcatttatt tcattttctt agaatagttt agtttattca
     4201 ttttatagtc acgaatgttt tatgattcta tatagggttg caaacaagca tttttcattt
     4261 tatgttaaaa caatttcagg tttacctttt attctgcttg tggtgacgcg tgtatccgcc
     4321 cgctcttttg gtcacccatg tatgctgcca aaccagatgt caacacagct acaacggcta
     4381 gtcggaatga acattcattc cgtgacctag gatgtgacgg aatgaaggtt cattccggac
     4441 tctagataag cacggaatga actttcattc cgctgaagct tgtcaatcgg aatgaaggtt
     4501 cattccggct agtcggaatg aacattcatt ccgcgaccta ggatgtgacg gaatgaaggt
     4561 tcattccgga ctctagataa gcacggaatg aactttcatt ccgctgaagc ttgtcaatcg
     4621 gaatgaaggt tcattccggc tatctccccg gaaactgtgg ccttttctgg cacacatgat
     4681 ctccacgatt tcaacatata aatagctttt gataatggca atattaatca aatttatttt
     4741 acttctttct tgtaacatct ctcttgtaat cccttattcc ttctagctat ttttcataaa
     4801 aaaccaagca actgcttatc aacacacaaa cactatggtg agcaagggcg aggaggataa
     4861 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtggacgg
     4921 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     4981 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     5041 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     5101 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     5161 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     5221 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacta tgggctggga
     5281 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     5341 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     5401 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     5461 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     5521 cggcatggac gagctgtaca agtaaatcct aactcgaggc caattggtgc ggcaattgat
     5581 aataacgaaa atgtctttta atgatctggg tataatgagg aattttccga acgtttttac
     5641 tttatatata tatatacatg taacatatat tctatacgct atagagaaag gaaatttttc
     5701 aattaaaaaa aaaatagaga aagagtttca cttcttgatt atcgctaaca ctaatggttg
     5761 aagtactgct actttaattt tatgctgagc agttacagag atgttacgaa ccactagtgc
     5821 actgcagtac acggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag
     5881 aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa
     5941 catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacgtgc aggaaacgaa
     6001 gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg
     6061 ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg
     6121 ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac
     6181 taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa
     6241 aggcattatc cgccaagtac aattttttac tcttcgagga cagaaaattt gctgacattg
     6301 gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca
     6361 ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag
     6421 aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct
     6481 ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt
     6541 ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt
     6601 tgattatgac acccggtgtg ggtttagatg acaagggaga tgcattgggt caacagtata
     6661 gaaccgtgga tgatgtggtt tctacaggat ctgacattat tattgttgga agaggactat
     6721 ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag
     6781 catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta
     6841 tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc gagttatcgt
     6901 gttgcatctg cggctttaaa ttgatgtatc tcatcgcagg cacgggcagt acagtgccct
     6961 gagcgtaggg aaaaatgaaa aaaaggatgt aacttttaac ataattccag cacgcagcga
     7021 ttgggtataa tgaagattgt taagttcaac aacatgtgaa attcttagct ccgcaacttg
     7081 cttgaattat gagctctaag attcaagaag taaataggtc atataatgga atgatagtag
     7141 tacttgataa aggaaccatg tgatcttacg ttgatatgaa aataattctc tcacagaagt
     7201 cagtttgtat gactaacata gagagtaatt gcttttcgga taaaatcgca cctaacaaag
     7261 attacctttt gtaaagcctc cagaacagct atgaatgttc gttattctac gcactttctt
     7321 tcatacatct tgatccgcct tttttgatgt aggttttaac gcggcaaaac atattaattt
     7381 ttctgatttg gaaaaataaa gagttagttc tgccccgagc ggccgcgtgt tacaaccaat
     7441 taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     7501 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     7561 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     7621 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     7681 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
     7741 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     7801 tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac
     7861 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     7921 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     7981 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     8041 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     8101 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     8161 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     8221 aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg
     8281 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
     8341 caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg
     8401 ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8461 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     8521 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     8581 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     8641 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     8701 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     8761 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     8821 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     8881 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     8941 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     9001 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     9061 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     9121 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     9181 tgcggccgcc aattctaatt gtatcgagat cacttttcgt gatccgctaa tcagcgacgg
     9241 tcacattagg tttgccaagt cagggtatga accatacgat cagttttcgt gaacctggta
     9301 cgtatattgt ggcgtttgtg tatattttca ttctttgaca acaatcaata ccaacctcaa
     9361 ataggaaaag taataagttt ggcgttacac cccaaaagac gccaaacgga tcgaacttac
     9421 tcaatagcaa ttagcgagac aaaacctacg ttaagacctg taaccgattt atcaaagcac
     9481 tctgcggttc tttcttggga atattacctg gacattttgt gccctcaaga aacgaggctc
     9541 tacgagcctg ttggagcccc tcagacatta gccgccacga atcaaacttt ttacgcgatt
     9601 cggcccaaat cagtttctca cagatcattc gtagagtgaa aaagcacatc gattatttga
     9661 tacccctttg ggttaattac tgttgaggtc tttgccgtaa gttccatcgc tgtccctgaa
     9721 ttcgcatcta gactgaactg gccgataa
//
