LOCUS       sequence-317760-        8834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pYES2 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 8834)
  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 8834)
  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: pEFEX1-A2aGH
FEATURES             Location/Qualifiers
     source          1..8834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        638..981
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     RBS             1446..1454
                     /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)"
     CDS             join(2574..2642,2643..3434)
                     /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     complement(2792..2811)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3605..4193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4094..4113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4347..4364
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4441..4688)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     4654..4672
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(4759..4788)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(join(4801..5511,5512..5514,5515..5517))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4801..5511,5512..5514,5515..5517))
                     /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(4843..4864)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5192..5211
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5451..5472
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6909..6928)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6910..6928)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6960..7401)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(6966..6989)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    7284..7401
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     rep_origin      7423..7936
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7510..7529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7720..7741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      7947..8824
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tgctattgaa gtgcaagatg gaaacgcaga aaatgaaccg gggatgcgac gtgcaagatt
       61 acctatgcaa tagatgcaat agtttctcca ggaaccgaaa tacatacatt gtcttccgta
      121 aagcgctaga ctatatatta ttatacaggt tcaaatatac tatctgtttc agggaaaact
      181 cccaggttcg gatgttcaaa attcaatgat gggtaacaag tacgatcgta aatctgtaaa
      241 acagtttgtc ggatattagg ctgtatctcc tcaaagcgta ttcgaatatc attgagaagc
      301 tgcagcgtca catcggataa taatgatggc agccattgta gaagtgcctt ttgcatttct
      361 agtctctttc tcggtctagc tagttttact acatcgcgaa gatagaatct tagatcacac
      421 tgcctttgct gagctggatc aatagagtaa caaaagagtg gtaaggcctc gttaaaggac
      481 aaggacctga gcggaagtgt atcgtacagt agacggagta tactaggtat agtctatagt
      541 ccgtggaatt aattctcatg tttgacagct tatcatcgat aagagctgac ctagatctgt
      601 ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac atggaggccc agaataccct
      661 ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc cgtacattta
      721 gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc gcacggcgcg
      781 aagcaaaaat tacggctcct cgctgcagac ctgcgagcag ggaaacgctc ccctcacaga
      841 cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc
      901 actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca gttctcacat
      961 cacatccgaa cataaacaac ccggccgatg attttcaatc cggtcatatc aaatcacaag
     1021 ttgagccatt atattcatgt tttttgtaca ttcaccacgt tctgcatatt aggtactgaa
     1081 acacgtcagg ctataacggc gctttccacc tacactccgg ccttcgttac tgctccgacc
     1141 gttctctggt ctaattgttc atcctgcatg ctaatgggga ttatgcaatc tttaaatgct
     1201 tacacttgga tgaaagatca ccaagtcttg tttttaggtg taactactgg gtactgtggt
     1261 gccctgtctt ccttttctag tatgctgcta gagatgttcg aacactctac aaacttaact
     1321 aacggtaata tcgctaacca cacaaaactg ccgaacagag cttacggtat aatggagttt
     1381 ttatctgtct tgctcgttca tcttatggtt tccatgggta gtcttatttt cggtaggcaa
     1441 cttggtaagg aggttattgt tgcttatgga tctagttcgt tttcgaagcc atatacccct
     1501 ccctcagaca ctgtaaagga aaatgctggt gatgttgata cacaagaaat ggaaaaaaat
     1561 attctagagt ttaaattcaa aactccagca ccatttttta agaaattttt tgatattgtg
     1621 gataaactcg cgtatgcact agcttttcca ttaattattt tgtttgtggt gctgtgtgcg
     1681 tattatgaga actattcgag gggcaagtgg acactaccat gtctatttgg aattttcgct
     1741 ggtttcctaa gatattggtt ggcagaaatg tttaataaaa caaacaaaaa gtttccattg
     1801 ggtactttct tggcaaacgt tttcgccact ttattgattg gcatatttac catggtgcaa
     1861 cgtggtaaaa aacacttttc tacggacgtt ccaattgtga actcgttgaa ttcatgtcat
     1921 attgtttctg cgttaatatc cggattttgc ggcactttga gtaccattag cacgtttatc
     1981 aatgaagggt ataagctatc atttattaac atgcttattt attatactgt ttcgattgca
     2041 atttcatatt gcttattggt gataactctc ggatcctatg cttggactag aggattgacc
     2101 aacccgattt gttagcttaa gagttgaaga catgggtatc tattactgta cgggttctta
     2161 ctatggtatg gactactggg gtcaaggaac ctcagtcacc gtctcctgat aacaaaagct
     2221 tatttctgaa gaagacttgt aatagctcga gatctgataa caacagtgta gatgtaacaa
     2281 aatcgacttt gttcccactg tacttttagc tcgtacaaaa tacaatatac ttttcatttc
     2341 tccgtaaaca acatgttttc ccatgtaata tccttttcta tttttcgttc cgttaccaac
     2401 tttacacata ctttatatag ctattcactt ctatacacta aaaaactaag acaattttaa
     2461 ttttgctgcc tgccatattt caatttgtta taaattccta taatttatcc tattagtagc
     2521 taaaaaaaga tgaatgtgaa tcgaatccta agagaattga cgaggagtag agtatgagta
     2581 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2641 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2701 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2761 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     2821 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2881 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2941 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3001 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3061 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3121 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3181 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3241 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3301 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3361 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3421 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3481 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3541 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3601 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3661 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3721 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     3781 acttcaagaa ctctgtagca ctgcctacat acctcgctct gctaatcctg ttaccagtgg
     3841 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3901 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3961 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4021 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4081 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4141 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4201 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4261 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4321 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4381 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg gccgcagctt
     4441 gcaaattaaa gccttcgagc gtcccaaaac cttctcaagc aaggttttca gtataatgtt
     4501 acatgcgtac acgcgtctgt acagaaaaaa aagaaaaatt tgaaatataa ataacgttct
     4561 taatactaac ataactataa aaaaataaat agggacctag acttcaggtt gtctaactcc
     4621 ttccttttcg gttagagcgg atgtgggggg agggcgtgaa tgtaagcgtg acataactaa
     4681 ttacatgatg cggccctcta gatgcatgct cgagccgtac agtaatagat acccatgtct
     4741 tcaactctta agttattaat gatgatgatg atgatgatga tgatgatgtg atgacacgtg
     4801 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     4861 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg
     4921 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     4981 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     5041 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     5101 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     5161 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     5221 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     5281 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt
     5341 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     5401 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     5461 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatccg
     5521 cggggacact cctgctccat cctgggccag ggggtcatct aggccagggg gctctgggca
     5581 cactcccttg agctcatggc taaggagctc cacgtctggg aggcccgtgt tcccctggga
     5641 ctcttgggca ctccctccac tcaccagccc cagggcgtag ccattgggcc tccgctcagg
     5701 gtggggagca ctgccgttgg cccacactcc tggcgggtgg ccgttgagac ggaggctgac
     5761 ctgctctccg tcactgccat gagctgccaa gacccgggca ctggtgccag ctgccttgaa
     5821 aggttcttgc tgcctcagga cgtggctgcg aatgatcttg cggaaggtct ggcggaactc
     5881 gcggatacgg taggcgtaga tgaagggatt cacaaccgaa ttggtgtggg agaggacgat
     5941 ggccaggtac atgagccaga gaggggcgtg gctgcagtcg gggcagaaga aagtgaagca
     6001 gttgatgatg tgtaggggca gccagcagag ggcaaagagc cccacaatga tggccagtga
     6061 cttggcagca tggacctcct tctgcagtgt ggaccgtgcc cgctcccccg gcagaggctg
     6121 gctctccatc tgcttcagct gtcgtcgcgc cgccaggaag atccgcaaat agacacccag
     6181 catgagcagc aggggcacca gcacacaggc aaagaagttg aagtacacca tgtagttcat
     6241 ggggaccaca tcctcaaaga gacaggccac ttggccctcc ccgcagccct gggagtggtt
     6301 cttgccctcc tttggctgac cgcagttgtt ccaacctagc atgggagtca ggccgatggc
     6361 aaacgacagc acccagcaga tggcaatgat gcccttagcc ctcgtgccgg tcaccaagcc
     6421 attgtaccgg agcgggatgc ggatggcaat gtagcggtca atggcgatgg ccaggagact
     6481 gaagatggag ctctgcgtga ggaccaggac gaagcaggca atgaagaggc agccgtggca
     6541 ggcagcgcag aacccggtgc tgatggtgat ggcaaagggg atggcgagca cacccactgc
     6601 gatgtcggcc gccgccagtg acaccacaaa gtagttggtg acgttctgca ggttgctgtt
     6661 gagccacacg gcccagcaca ccagcacatt gcccaggatg gccagcacag caatggccag
     6721 ctccaccgtg atgtacaccg aggagcccat gatgggcatg acgtccggcc gagcttctct
     6781 cttgtccaaa gagccttctg cagcggaacc gacggtagta gaaataaccg gttgagctag
     6841 agccaagggc aaagcagcga agatagccaa caagacaatc aaaaccttca tgatccaagc
     6901 ttaatattcc ctatagtgag tcgtattaca gctgctagta gtccgatccg gggttttttc
     6961 tccttgacgt taaagtatag aggtatatta acaatttttt gttgatactt ttattacatt
     7021 tgaataagaa gtaatacaaa ccgaaaatgt tgaaagtatt agttaaagtg gttatgcagt
     7081 ttttgcattt atatatctgt taatagatca aaaatcatcg cttcgctgat taattacccc
     7141 agaaataagg ctaaaaaact aatcgcatta tcatcctatg gttgttaatt tgattcgttc
     7201 atttgaaggt ttgtggggcc aggttactgc caatttttcc tcttcataac cataaaagct
     7261 agtattgtag aatctttatt gttcggagca gtgcggcgcg aggcacatct gcgtttcagg
     7321 aacgcgaccg gtgaagacga ggacgcacgg aggagagtct tccttcggag ggctgtcacc
     7381 cgctcggcgg cttctaatcc gtggactagt ggatcgatcc ccacgcgccc tgtagcggcg
     7441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     7501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     7561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     7621 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     7681 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     7741 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     7801 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     7861 tattaacgct tacaatttaa atatttgctt atacaatctt cctgtttttg gggcttttct
     7921 gattatcaac cggggtggag cttcccattg cgaataccgc ttccacaaac attgctcaaa
     7981 agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc atccaccttt
     8041 cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat ctctagtatt
     8101 actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga aaacaatacg
     8161 aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa ccgttcataa
     8221 ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag tatgcgcaat
     8281 ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc acccgcagct
     8341 tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga agagaaaata
     8401 gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag ttatcaagag
     8461 actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt gttagaaaaa
     8521 tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct ttgttggtaa
     8581 aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
     8641 aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag attcttcgtt
     8701 ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg
     8761 tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc acagatgctt
     8821 cgttaacaaa gata
//
