LOCUS       sequence-429384-        7781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative fluorescent reporter for amino acid biosynthesis sensing
            pathways, comprising GFP driven by the ARG8 promoter within the Easy
            clone vector, requires NotI digestion for integration in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7781)
  AUTHORS   Paukstyte J, Tena EC, Saarikangas J
  TITLE     A dual reporter system for intracellular and extracellular amino 
            acid sensing in budding yeast.
  JOURNAL   Mol Biol Cell. 2025 Apr 2:mbcE24040162. doi: 
            10.1091/mbc.E24-04-0162.
  PUBMED    40172974
REFERENCE   2  (bases 1 to 7781)
  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: "Mol Biol 
            Cell. 2025 Apr 2:mbcE24040162. doi: 10.1091/mbc.E24-04-0162."
COMMENT     Sequence Label: pCfB2188-PrARG8-GFP::URA3
FEATURES             Location/Qualifiers
     source          1..7781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      12..201
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    249..282
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1734..1767
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1810..1829)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1811..1829)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1839..1855)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1848..1870)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2448..2481)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2951..2967)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3066..3083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3237..3825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3317..3336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3947..4738,4739..4807))
                     /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     4570..4589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4808..4899)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4941..4960
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4966..4985
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5192..5225
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(5835..6000)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5921..5945
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             7063..7779
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 cgcgtgcatt catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
       61 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
      121 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
      181 aaggttttgg gacgctcgaa gatcgcgtca gctgaagctt cgtacgctgc aggtcgacaa
      241 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtcta gagatcccaa
      301 tacaacagat cacgtgatct ttttgtaaga tgaagttgaa gtagtgttgc accgtgccaa
      361 tgcaggtggc tattagatta aatatgtgat ttgttctatt aagtttcctg tataattaat
      421 ggggagcgct gattctcttt tggtacgctt cccatccagc atttctgtat ctttcacctt
      481 caaccttagg atctctaccc ttggcgaaaa gtcctctgcc aacaatgatg atatctgatc
      541 caccacttac aacttcgtcg acggttctgt actgctgacc caatgcatcg cctttgtcgt
      601 ctaaacctac acctggggtc atgattagcc aatcaaaccc ttcttctctt cctcccatat
      661 cgttctgagc aatgaaccca ataacgaaat ctttatcact ctttgcaata tcaacggtac
      721 ccttagtata ttcaccgtgt gctagagaac ccttggaaga taattcagca agcatcaata
      781 atccccttgg ttctttggtg acctcttgcg caccttgttt caagccagca acaataccag
      841 caccagtaac cccgtgggcg ttggtgatat cagaccattc tgcgatacgg taaacgcccg
      901 atgtatattg taatttgact gtgttaccga tatcggcgaa ttttctgtcc tcaaatatca
      961 agaacttgta tttctctgcc aatgctttca atggaacgac agtaccctca taactgaaat
     1021 catccaagat atcaacgtgt gttttcaaaa ggcaaatgta tggacccaac gtttcaacaa
     1081 gtttcaatag ctcatcagtc gaacgaacgt caagagaagc acacaaattg gttttctttt
     1141 catccattaa acgtaaaagt ttcgatgcaa ccggacttgc atgagtctca gctctactgg
     1201 tatatgattt tgtggacatg gtgcaactaa ttgacgggag tgtattgacg ctggcgtact
     1261 ggctttcaca aaatggccca atcacaacca catcttagat agttgaaatg actttagata
     1321 acatcaattg agatgagctt aatcatgtca aagctaaaag tgtcaccatg aacgacaatt
     1381 cttaagcaaa tcacgtgata tagatccacg aataaccacc atttgatgct cgaggcaagt
     1441 aatgtgtgta aaaaaatgcg ttaccaccat ccaatgcaga ccgatcttct acccagaatc
     1501 acatatattt atgtaccgag tacctttttt ctatcttcca attgcttctc ccatatgatt
     1561 gtctccgtaa gctcgaaatt tctaagttgg attttaatct tcacgcagga tgacagttcg
     1621 atgagcttct gaggagtgtt tagaacataa tcatttatcc atggtctatc tcttcttgtc
     1681 gctttttctc ctcgatagaa cctaaataaa acgagctctc gagaaccctt aatataactt
     1741 cgtataatgt atgctatacg aagttattag gtgatatcag atccactagt ggcctatgca
     1801 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     1861 cgtgactggg aaaaccctgg cgttacccct gcaggactag tgctgaggca ttaatcctgc
     1921 ataatcggcc tcacagaggg atcccgttac ccatctatgc tgaagattta tcatactatt
     1981 cctccgctcg tttctttttt cagtgaggtg tgtcgtgaaa gaaaacccac aattaaactt
     2041 tcaacaaccg ggcgactagg aagagagtag tgggcgcgga tgacgaaggc taaggtcact
     2101 tcttcgtttc ctttattggg gtttccgtgt agccttcccc tgaatagtgt gggacgtttt
     2161 atgagaagcc gtaagaaata ggcaaattga gttatgacaa gtagacatga tgccgcagcc
     2221 ttgcctgact ttacgtctcc ttcatgaata agtttttcta tcgagttctt ttcctttttt
     2281 cgccttaatt agctcaatta agcctgtcct cactactttt ctttttctta tcggctttgt
     2341 gccacaccta accttcgaat gctgttttat tccgttctta catgggatgg taatgccttg
     2401 gcgagattaa gacctcagcg cggccgcaaa tttaaataaa atgaagtgaa gttcctatac
     2461 tttctagaga ataggaactt ctatagtgag tcgaataagg gcgacacaaa atttattcta
     2521 aatgcataat aaatactgat aacatcttat agtttgtatt atattttgta ttatcgttga
     2581 catgtataat tttgatatca aaaactgatt ttccctttat tattttcgag atttattttc
     2641 ttaattctct ttaacaaact agaaatattg tatatacaaa aaatcataaa taatagatga
     2701 atagtttaat tataggtgtt catcaatcga aaaagcaacg tatcttattt aaagtgcgtt
     2761 gcttttttct catttataag gttaaataat tctcatatat caagcaaagt gacaggcgcc
     2821 cttaaatatt ctgacaaatg ctctttccct aaactccccc cataaaaaaa cccgccgaag
     2881 cgggttttta cgttatttgc ggattaacga ttactcgtta tcagaaccgc ccagggggcc
     2941 cgagcttaag actggccgtc gttttacaac acagaaagag tttgtagaaa cgcaaaaagg
     3001 ccatccgtca ggggccttct gcttagtttg atgcctggca gttccctact ctcgccttcc
     3061 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3121 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3181 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3241 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3301 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3361 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3421 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3481 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3541 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3601 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgggctaac
     3661 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3721 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3781 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3841 ttttctacgg ggtctgacgc tcagtggaac gacgcgcgcg taactcacgt taagggattt
     3901 tggtcatgag cttgcgccgt cccgtcaagt cagcgtaatg ctctgcttac caatgcttaa
     3961 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4021 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga
     4081 taccgcgaga accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa
     4141 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4201 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg
     4261 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4321 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4381 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4441 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4501 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4561 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4621 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4681 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4741 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4801 tactcatatt cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4861 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggtcagt gttacaacca
     4921 attaaccaat tctgaacatt atcgcgagcc catttatacc tgaatatggc tcataacacc
     4981 ccttgtttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
     5041 gtgaaacgcc gtagcgccga tggtagtgtg gggactcccc atgcgagagt agggaactgc
     5101 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgcc cgggctaatt
     5161 atggggtgtc gcccttattc gactctatag tgaagttcct attctctaga aagtatagga
     5221 acttctgaag tggggattta aatgcggccg cgctgagggt ttaatcgtct atgaggagac
     5281 tgttagttgg atatcagtaa tgagacgaaa aagctcgaaa tgaatggata tattcttttt
     5341 gctactggca actgttgaat atttaatgtt aaaacaaact aactgaggta tattcgtatc
     5401 tgtatgtaca catatactat atacaggaaa agataagcaa gagagaggat atcaactacg
     5461 agagcgatcg attatatatc aaaagctgtc cgctttgcca cccataatcg gcgcttagtt
     5521 tcggagttca atcataattc taccacctta cactcaactt actctttaac tcctatagta
     5581 taatatcgcc actgacccca tattaaaaaa tttttttgct cgatcttcta tcctctttag
     5641 gttaattgtc gctgttattg tctagatttt ttctcggaga tggcgcatct atttgccgtc
     5701 aaaagatcct ctcataccat attaagtaaa ttgcctccat ttctttttcc tcgggcagag
     5761 aaactcgcag gcaacttgct ctcgaagtgg tcacgtatta agtcctcagc gagctcgcat
     5821 ggaatgcgtg cgatgagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa
     5881 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta
     5941 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc
     6001 ttatttagaa gtgtcaacaa cgtatctacc aacggaatgc gtgcgattcc tgtagcaaga
     6061 atgtgtcaca cttttacaac tatttctaga aataaccagc tactgcttat tggaggtaga
     6121 aaggcaccac accaaggtct ctctgataat tggatatttg atatgaaaac aagagagtgg
     6181 tcgatgatca agagtttatc acacacgaga ttcagacata gtgcatgtag cttaccggat
     6241 ggaaacgtcc tgatccttgg tggggttaca gaaggacctg ccatgctgct atacaatgtc
     6301 accgaagaaa tcttcaaaga cgtaactcca aaagatgaat tcttccaaaa ttcgctagta
     6361 tcagcgggtc tagaatttga tccggtttcc aaacaaggca taatattggg tggtggattt
     6421 atggatcaaa caactgtctc ggacaaggca attattttca aatatgatgc ggaaaatgct
     6481 actgaaccaa ttacggtgat taagaagctc cagcacccct tatttcaacg ttatggatcc
     6541 cagataaaat acattactcc aagaaagcta ttaatagtcg gtggaacaag tccttccggg
     6601 ctatttgacc ggaccaactc tataatcagc cttgatccgc tgagtgagac gctaacatca
     6661 attcccatat caagacgcat atgggaagat cactcactaa tgcttgctgg atttagtttg
     6721 gtgtccactt ctatgggcac catacatatc attggaggcg gtgccacttg ctacggcttt
     6781 ggatcagtga ctaatgtagg tcttaagctc atagcaatcg cgaaataatg ctcctatcgg
     6841 gattcgaatg gttgccagct cgctatgtga ctcacttaaa gtacatgatg cgtcattgtg
     6901 cactttttta cgaaataatg gcctttttcc gctctattta aaagcgtgaa aaaaaaattg
     6961 caacatgaag aaaattcgac tcttaagtgc aagtgactgc gaacattttt ttcgtttgtt
     7021 agaataattc aagaatcgct accaatcatc tgtcataaaa caatgtctaa aggtgaagaa
     7081 ttattcactg gtgttgtccc aattttggtt gaattagatg gtgatgttaa tggtcacaaa
     7141 ttttctgtct ccggtgaagg tgaaggtgat gctacttacg gtaaattgac cttaaaattt
     7201 atttgtacta ctggtaaatt gccagttcca tggccaacct tagtcactac tttcggttat
     7261 ggtgttcaat gttttgcgag atacccagat catatgaaac aacatgactt tttcaagtct
     7321 gccatgccag aaggttatgt tcaagaaaga actatttttt tcaaagatga cggtaactac
     7381 aagaccagag ctgaagtcaa gtttgaaggt gataccttag ttaatagaat cgaattaaaa
     7441 ggtattgatt ttaaagaaga tggtaacatt ttaggtcaca aattggaata caactataac
     7501 tctcacaatg tttacatcat ggctgacaaa caaaagaatg gtatcaaagt taacttcaaa
     7561 attagacaca acattgaaga tggttctgtt caattagctg accattatca acaaaatact
     7621 ccaattggtg atggtccagt cttgttacca gacaaccatt acttatccac tcaatctgcc
     7681 ttatccaaag atccaaacga aaagagagac cacatggtct tgttagaatt tgttactgct
     7741 gctggtatta cccatggtat ggatgaattg tacaaatgaa t
//
