LOCUS       sequence-446753-        9859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing a cytosolic cAMP sensor (mScarletI3-ApacH-StayGold2) in 
            Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9859)
  AUTHORS   Evans S, Tominaga M, Lu Z, Bandari NC, McDonnell L, Wang C, Speight 
            RE, Ishii J, Vickers CE, Peng B
  TITLE     Characterization of Fluorescent Reporters for Flow Cytometry-Based 
            Single-Cell Studies in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2025 Sep 19;14(9):3804-3812. doi: 
            10.1021/acssynbio.5c00432. Epub 2025 Sep 10.
  PUBMED    40927980
REFERENCE   2  (bases 1 to 9859)
  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"; doi: 
            "10.1021/acssynbio.5c00432"; journalName: "ACS Synth Biol"; date: 
            "2025-09-19- 19"; volume: "14"; issue: "9"; pages: "3804-3812"
COMMENT     Sequence Label: pILFPQ13BE5
FEATURES             Location/Qualifiers
     source          1..9859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1..271
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(536..552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(536..552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(549..571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    560..576
                     /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(584..590,591..608,609..614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    629..650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(767..784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(938..1526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1018..1037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1697..2488,2489..2557))
                     /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     2320..2339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2558..2662)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2730..2748
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2786..2808)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2908..2927
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3121..3143
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3135..3152
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3136..3152
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4456..4799
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      5192..5389
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(5596..5629)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5849..6262
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             9192..9842
                     /codon_start=1
                     /product="bright and highly photostable dimeric green 
                     fluorescent protein (Hirano et al., 2022)"
                     /label=StayGold
                     /note="engineered variant of a GFP from Cytaeis uchidae"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
ORIGIN
        1 attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc catcctttac
       61 gctaaaataa tagtttattt tattttttga atatttttta tttatatacg tatatataga
      121 ctattattta tcttttaatg attattaaga tttttattaa aaaaaaattc gctcctcttt
      181 taatgccttt atgcagtttt tttttcccat tcgatatttc tatgttcggg ttcagcgtat
      241 tttaagttta ataactcgaa aattctgcgt tcgttaaagc tttcgagaag gatattattt
      301 actagtagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa
      361 agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata
      421 agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt
      481 acccgggaat ctcggtcgta atgatttaaa tgcatgcaag cttggcgtaa tcatggtcat
      541 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
      601 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
      661 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
      721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
      781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     2701 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     2761 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2821 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2881 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2941 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     3001 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     3061 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     3121 cccagtcacg acgttgtaaa acgacggcca gtgaattcat ttaaattttt ttgattcggt
     3181 aatctccgaa cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg
     3241 catatgtagt gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac
     3301 aaaaacctgc aggaaacgaa gataaaacat gtccacaaaa tcatatacca gtagagctga
     3361 gactcatgca agtccggttg catcgaaact tttacgttta atggatgaaa agaagaccaa
     3421 tttgtgtgct tctcttgacg ttcgttcgac tgatgagcta ttgaaacttg ttgaaacgtt
     3481 gggtccatac atttgccttt tgaaaacaca cgttgatatc ttggatgatt tcagttatga
     3541 gggtactgtc gttccattga aagcattggc agagaaatac aagttcttga tatttgagga
     3601 cagaaaattc gccgatatcg gtaacacagt caaattacaa tatacatcgg gcgtttaccg
     3661 tatcgcagaa tggtctgata tcaccaacgc ccacggggtt actggtgctg gtattgttgc
     3721 tggcttgaaa caaggtgcgc aagaggtcac caaagaacca aggggattat tgatgcttgc
     3781 tgaattgtct tccaagggtt ctctagcaca cggtgaatat actaagggta ccgttgatat
     3841 tgcaaagagt gataaagatt tcgttattgg gttcattgct cagaacgata tgggaggaag
     3901 agaagaaggg tttgattggc taatcatgac cccaggtgta ggtttagacg acaaaggcga
     3961 tgcattgggt cagcagtaca gaaccgtcga cgaagttgta agtggtggat cagatatcat
     4021 cattgttggc agaggacttt tcgccaaggg tagagatcct aaggttgaag gtgaaagata
     4081 cagaaatgct ggatgggaag cgtaccaaaa gagaatcagc gctccccatt aattatacag
     4141 gaaacttaat agaacaaatc acatatttaa tctaatagcc acctgcattg gcacggtgca
     4201 acactacttc aacttcatct tacaaaaaga tcacgggatc tgttgtattg ggatctctag
     4261 gctgaagctt cgtacgctgc aggtcgacaa cccttaatgt tcttcattct acgcacggtc
     4321 cttcaattcg cttggcggag atctatgaca agagggtcga actcgcctaa gtcgtaattg
     4381 agtcagttac gctagggata acagggtaat atagctgttt agcttgcctc gtccccgccg
     4441 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     4501 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     4561 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     4621 ctgcagacct gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc
     4681 gcgcccctgt agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata
     4741 cttcctttta aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca
     4801 tgggtatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat cccaccgccg
     4861 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4921 agcgcgggga tctcatgctg gagttcttcg cccaccccgg gctcgatccc ctcgcgagtt
     4981 ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag tgcaaatccg
     5041 tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa ctgcaggagt
     5101 ggggaggcac gatggccgct ttggtcgacc cggacgggac gctcctgcgc ctgatacaga
     5161 acgaattgct tgcaggcatc tcatgatcag tactgacaat aaaaagattc ttgttttcaa
     5221 gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag
     5281 cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca
     5341 gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg
     5401 atactaacgc cgccatccag tgtcgaaaac gagctctcga gaacccttaa agttacgcta
     5461 gggataacag ggtaatatag gttcttcatt ctacgcacgg tccttcaatt cgcttggcgg
     5521 tctagaatga caagagggtc gaactcgcct aagtcgtaat tgagtccaga tccactagtg
     5581 gcctatctag acctaataac ttcgtatagc atacattata cgaagttata ttaagggttg
     5641 ctcgagaaag agctccagaa agcgaccacc caactttggc tgataatagc gtataaacaa
     5701 tgcatacttt gtacgttcaa aatacaatgc agtagatata tttatgcata ttacatataa
     5761 tacatatcac ataggaagca acaggcgcgt tggactttta attttcgagg accgcgaatc
     5821 cttacatcac acccaatccc ccacaagtga tcccccacac accatagctt caaaatgttt
     5881 ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa
     5941 aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta
     6001 cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa
     6061 aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt tttgattttt
     6121 ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca atttctcaag
     6181 tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga
     6241 aagcatagca atctaatcta agttttaatt acaaactcga gaaaggatcc aaaatggatt
     6301 ctaccgaagc tgtcatcaaa gagttcatga gattcaaggt tcacatggaa ggttctatga
     6361 acggtcacga atttgaaatt gaaggtgaag gtgagggtag accatacgaa ggtactcaaa
     6421 ctgcaaaact gaaagttaca aaaggtggtc cattgccatt ctcttgggat attttgtctc
     6481 cacaattcat gtacggttcc agagctttta tcaaacatcc agctgatatt ccagactact
     6541 ggaagcaatc atttcctgaa ggttttaagt gggagagagt catgattttt gaagatggtg
     6601 gtactgtctc tgttacccaa gatacttctt tggaagatgg cactttgatc tacaaggtta
     6661 agttgagagg tggtaatttt ccaccagatg gtccagttat gcagaaaaga actatgggtt
     6721 gggaagcctc tactgaaaga ttatatccag aagatgttgt cttgaagggc gatattaaga
     6781 tggctttgag attgaaagac ggtggtagat atttggctga tttcaagacc acttacaagg
     6841 ccaaaaagcc agttcaaatg ccaggtgctt ttaacattga taggaagttg gatattacca
     6901 gccacaacga agattacacc gttgttgaac aatacgaaag atccgttgct agacattcta
     6961 ctggtggttc aggtggttct ccagttggta ctcacgaaat ggaagaagaa ttggctgaag
     7021 ctgttgcttt gttgtctcaa agaggtccag acgctttgtt gacagttgct ttgagaaaac
     7081 caccaggtca aagaactgac gaagaactgg atttgatttt cgaagagttg ttgcatatta
     7141 aggccgttgc tcatttgtcc aactctgtta agagagaatt agctgccgtt ttgttgttcg
     7201 aaccacattc taaagctggt actgtcttat tttctcaagg tgacaaaggt acgagctggt
     7261 acattatttg gaagggttct gttaacgttg tcacccatgg taaaggtttg gttacaacat
     7321 tgcacgaagg tgacgatttt ggtgaattgg ctttggttaa tgatgctcca agagctgcta
     7381 ctatcatttt gagagaagat aactgccact tcttgagagt tgataagcaa gacttcaaca
     7441 ggatcatcaa ggatgttgaa gctaagacca tgagattgga agaacacggt aaagttgttt
     7501 tggttttgac cagagcttca caaggtgctg gtccatcaag accaccaact ccaggtagaa
     7561 atagatacac tgttatgtct ggtacgcccg aaaagatttt ggaattgcta ttggaagcta
     7621 tgggtccaga ttcttctgct catgatccaa ctgaaacttt cttgtccgat ttcttgttga
     7681 cccacagagt tttcatgcca tctgctcaat tatgtgcagc tttgttgcat catttccatg
     7741 ttgaacctgc tggtggtagt gaacaagaaa gatcaactta cgtctgcaac aagagacagc
     7801 aaatcttgag attggtttct caatgggttg ccttgtatgg ttctatgttg catactgatc
     7861 cagttgctac atccttcttg caaaagttgt ctgatttggt tggtagagac accagattgt
     7921 ctaacttgtt gagagaacaa tggccagaaa gacgtagatg tcatagattg gaaaatggtt
     7981 gtggtaacgc ttctccacaa atgaaggcta gaaatttgcc tgtttggttg ccaaatcaag
     8041 atgaaccatt gccaggttct tcatgcgcta ttcaagttgg tgacaaggtt ccatacgata
     8101 tctgtagacc agatcattct gttttgacat tgcagttgcc agttactgct tctgttagag
     8161 aagttatggc tgctttggct caagaggatg gttggactaa gggtcaagtt ttggttaagg
     8221 ttaattctgc tggtgacgcc attggtttac aaccagatgc tagaggtgtt gctacttctt
     8281 tgggtttgaa cgaaaggttg tttgtcgtta atccacaaga agcccacgaa ttgattccac
     8341 atcctgatca attaggtcca actgttggtt ctgctgaagg tttggatttg gtttctgcaa
     8401 aagatttggc tggtcagttg actgatcatg attggtcttt gttcaactcc atccaccaag
     8461 ttgaattgat ccattatgtt ttgggtccac agcacttgag agatgttact acagctaatt
     8521 tggagaggtt catgagaagg ttcaacgaat tgcaatattg ggttgctacc gaattgtgtt
     8581 tgtgtccagt tccaggtcca agagcacaat tattgagaaa gttcattaag ttggctgccc
     8641 acttgaaaga acagaagaat ttgaactctt tcttcgccgt tatgttcggc ttgtctaatt
     8701 cagctatttc cagattggct catacctggg aaagattgcc acataaggtt agaaagttgt
     8761 acagtgcatt ggaaagacta ttggacccat cttggaatca tagagtttac agattagctc
     8821 tggctaagtt gtctccacca gttattcctt ttatgccatt attggcagct gatatggctg
     8881 caattcatga aggtaatcac actttggtcg agaacttgat caacttcgaa aagatgagaa
     8941 tgatggctag agctgcaaga atgttacatc actgtagatc ccataatcct gttccattgt
     9001 ctccattgag atcaagagtt tctcacttgc atgaagattc ccaagttgct agaatttcca
     9061 cctgttctga acaatctttg tctactagaa gtccagcttc tacttgggct tatgttcaac
     9121 agttgaaggt tattgacaac cagagggaat tgtccagatt gagtagagaa ttggaaccag
     9181 cttctgagtt gatggcttct actccattca agtttcagtt gaagggtact attaacggca
     9241 agtcttttac cgttgaaggt gaaggtgagg gtaattctca tgaaggttct cataagggca
     9301 aatacgtatg tacttctggc aaattgccaa tgtcttgggc tgctttgggt acttcttttg
     9361 gttatggtat gaagtattac acgaagtacc cctctggttt gaagaattgg tttcatgaag
     9421 ttatgcctga gggtttcact tacgatagac atattcagta caaaggtgac ggttccattc
     9481 atgccaaaca tcaacatttt atgaagaacg gcacctacca taacatcgtt gagtttactg
     9541 gtcaggactt caaagaaaac tctccagttt tgactggtga catgaatgtt tctttgccaa
     9601 acgaagttca acacatccca agagatgatg gtgttgaatg tccagttact ttgttgtacc
     9661 cactgttgtc tgataagtct aagtgtgttg aagctcacca aaacactatc tgtaagccat
     9721 tgcataatca accagctcca gatgttccat accattggat tagaaaacag tacacccaat
     9781 ccaaggatga cactgaagaa agagatcata tctgccaatc tgaaaccttg gaagctcatt
     9841 tgtaaagatc tgatcttaa
//
