LOCUS       sequence-317845-        7594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pYC2/CT derivative reporter plasmid encoding Ashbya gossypii 
            PTEF1-Saccharomyces cerevisiae FEX1-MFaT; as well as PGAL1-Homo 
            sapiens ADORA2A-EGFP-10HIS-MFaT..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7594)
  AUTHORS   Yoo JI, Seppala S, O'Malley MA
  TITLE     Engineered fluoride sensitivity enables biocontainment and selection
            of genetically-modified yeasts.
  JOURNAL   Nat Commun. 2020 Oct 29;11(1):5459. doi: 10.1038/s41467-020-19271-1.
  PUBMED    33122649
REFERENCE   2  (bases 1 to 7594)
  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: "Nat 
            Commun."; date: "2020-10-29"; volume: "11(1):5459. doi"; pages: " 
            10.1038/s41467-020-19271-1"
COMMENT     Sequence Label: pCFEX1-A2aGH
FEATURES             Location/Qualifiers
     source          1..7594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        153..594
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    153..270
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     565..588
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2037..2039,2040..2042,2043..2753)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2037..2039,2040..2042,2043..2753)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2082..2103)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2343..2362)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2690..2711
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2766..2795
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2864..2905
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2915..2932
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2966..3213
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2982..3000)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3290..3307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3461..4049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3541..3560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4220..5011,5012..5080))
                     /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     4843..4862
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     RBS             6200..6208
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(6673..7016)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     misc_feature    7085..7589
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
       61 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
      121 acagggcgcg tggggatcga tccactagtc cacggattag aagccgccga gcgggtgaca
      181 gccctccgaa ggaagactct cctccgtgcg tcctcgtctt caccggtcgc gttcctgaaa
      241 cgcagatgtg cctcgcgccg cactgctccg aacaataaag attctacaat actagctttt
      301 atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca aatgaacgaa
      361 tcaaattaac aaccatagga tgataatgcg attagttttt tagccttatt tctggggtaa
      421 ttaatcagcg aagcgatgat ttttgatcta ttaacagata tataaatgca aaaactgcat
      481 aaccacttta actaatactt tcaacatttt cggtttgtat tacttcttat tcaaatgtaa
      541 taaaagtatc aacaaaaaat tgttaatata cctctatact ttaacgtcaa ggagaaaaaa
      601 ccccggatcg gactactagc agctgtaata cgactcacta tagggaatat taagcttgga
      661 tcatgaaggt tttgattgtc ttgttggcta tcttcgctgc tttgcccttg gctctagctc
      721 aaccggttat ttctactacc gtcggttccg ctgcagaagg ctctttggac aagagagaag
      781 ctcggccgga cgtcatgccc atcatgggct cctcggtgta catcacggtg gagctggcca
      841 ttgctgtgct ggccatcctg ggcaatgtgc tggtgtgctg ggccgtgtgg ctcaacagca
      901 acctgcagaa cgtcaccaac tactttgtgg tgtcactggc ggcggccgac atcgcagtgg
      961 gtgtgctcgc catccccttt gccatcacca tcagcaccgg gttctgcgct gcctgccacg
     1021 gctgcctctt cattgcctgc ttcgtcctgg tcctcacgca gagctccatc ttcagtctcc
     1081 tggccatcgc cattgaccgc tacattgcca tccgcatccc gctccggtac aatggcttgg
     1141 tgaccggcac gagggctaag ggcatcattg ccatctgctg ggtgctgtcg tttgccatcg
     1201 gcctgactcc catgctaggt tggaacaact gcggtcagcc aaaggagggc aagaaccact
     1261 cccagggctg cggggagggc caagtggcct gtctctttga ggatgtggtc cccatgaact
     1321 acatggtgta cttcaacttc tttgcctgtg tgctggtgcc cctgctgctc atgctgggtg
     1381 tctatttgcg gatcttcctg gcggcgcgac gacagctgaa gcagatggag agccagcctc
     1441 tgccggggga gcgggcacgg tccacactgc agaaggaggt ccatgctgcc aagtcactgg
     1501 ccatcattgt ggggctcttt gccctctgct ggctgcccct acacatcatc aactgcttca
     1561 ctttcttctg ccccgactgc agccacgccc ctctctggct catgtacctg gccatcgtcc
     1621 tctcccacac caattcggtt gtgaatccct tcatctacgc ctaccgtatc cgcgagttcc
     1681 gccagacctt ccgcaagatc attcgcagcc acgtcctgag gcagcaagaa cctttcaagg
     1741 cagctggcac cagtgcccgg gtcttggcag ctcatggcag tgacggagag caggtcagcc
     1801 tccgtctcaa cggccacccg ccaggagtgt gggccaacgg cagtgctccc caccctgagc
     1861 ggaggcccaa tggctacgcc ctggggctgg tgagtggagg gagtgcccaa gagtcccagg
     1921 ggaacacggg cctcccagac gtggagctcc ttagccatga gctcaaggga gtgtgcccag
     1981 agccccctgg cctagatgac cccctggccc aggatggagc aggagtgtcc ccgcggatgg
     2041 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2101 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2161 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2221 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2281 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2341 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2401 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2461 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2521 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2581 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2641 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2701 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcacgtgt
     2761 catcacatca tcatcatcat catcatcatc atcattaata acttaagagt tgaagacatg
     2821 ggtatctatt actgtacggc tcgagtctag agggcccttc gaaggtaagc ctatccctaa
     2881 ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagttta
     2941 aacccgctga tcctagaggg ccgcatcatg taattagtta tgtcacgctt acattcacgc
     3001 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     3061 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     3121 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     3181 aaggttttgg gacgctcgaa ggctttaatt tgcaagctgc ggccctgcat taatgaatcg
     3241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3301 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3361 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3421 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3481 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3541 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3601 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3661 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3721 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3781 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3841 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3901 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3961 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4021 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4081 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4141 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4201 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4261 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4321 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4381 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4441 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4501 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4561 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4621 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4681 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4741 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4801 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4861 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4921 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4981 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5041 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actctactcc tcgtcaattc
     5101 tcttaggatt cgattcacat tcatcttttt ttagctacta ataggataaa ttataggaat
     5161 ttataacaaa ttgaaatatg gcaggcagca aaattaaaat tgtcttagtt ttttagtgta
     5221 tagaagtgaa tagctatata aagtatgtgt aaagttggta acggaacgaa aaatagaaaa
     5281 ggatattaca tgggaaaaca tgttgtttac ggagaaatga aaagtatatt gtattttgta
     5341 cgagctaaaa gtacagtggg aacaaagtcg attttgttac atctacactg ttgttatcag
     5401 atctcgagct attacaagtc ttcttcagaa ataagctttt gttatcagga gacggtgact
     5461 gaggttcctt gaccccagta gtccatacca tagtaagaac ccgtacagta atagataccc
     5521 atgtcttcaa ctcttaagct aacaaatcgg gttggtcaat cctctagtcc aagcatagga
     5581 tccgagagtt atcaccaata agcaatatga aattgcaatc gaaacagtat aataaataag
     5641 catgttaata aatgatagct tatacccttc attgataaac gtgctaatgg tactcaaagt
     5701 gccgcaaaat ccggatatta acgcagaaac aatatgacat gaattcaacg agttcacaat
     5761 tggaacgtcc gtagaaaagt gttttttacc acgttgcacc atggtaaata tgccaatcaa
     5821 taaagtggcg aaaacgtttg ccaagaaagt acccaatgga aactttttgt ttgttttatt
     5881 aaacatttct gccaaccaat atcttaggaa accagcgaaa attccaaata gacatggtag
     5941 tgtccacttg cccctcgaat agttctcata atacgcacac agcaccacaa acaaaataat
     6001 taatggaaaa gctagtgcat acgcgagttt atccacaata tcaaaaaatt tcttaaaaaa
     6061 tggtgctgga gttttgaatt taaactctag aatatttttt tccatttctt gtgtatcaac
     6121 atcaccagca ttttccttta cagtgtctga gggaggggta tatggcttcg aaaacgaact
     6181 agatccataa gcaacaataa cctccttacc aagttgccta ccgaaaataa gactacccat
     6241 ggaaaccata agatgaacga gcaagacaga taaaaactcc attataccgt aagctctgtt
     6301 cggcagtttt gtgtggttag cgatattacc gttagttaag tttgtagagt gttcgaacat
     6361 ctctagcagc atactagaaa aggaagacag ggcaccacag tacccagtag ttacacctaa
     6421 aaacaagact tggtgatctt tcatccaagt gtaagcattt aaagattgca taatccccat
     6481 tagcatgcag gatgaacaat tagaccagag aacggtcgga gcagtaacga aggccggagt
     6541 gtaggtggaa agcgccgtta tagcctgacg tgtttcagta cctaatatgc agaacgtggt
     6601 gaatgtacaa aaaacatgaa tataatggct caacttgtga tttgatatga ccggattgaa
     6661 aatcatcggc cgggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt
     6721 ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc
     6781 tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg
     6841 caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg
     6901 gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat
     6961 gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg
     7021 gccgggtgac ccggcgggga cgaggcaagc taaacagatc taggtcagct gatgggtcct
     7081 tttcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt
     7141 aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta
     7201 aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag
     7261 gtattaatgc cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg
     7321 attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct
     7381 aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt
     7441 tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa
     7501 aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg
     7561 aggcgcgtgt aagttacagg caagcgatcc gtcc
//
