LOCUS       sequence-317791-        8533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pITY4 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 8533)
  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 8533)
  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: pIFEX-A2aGH
FEATURES             Location/Qualifiers
     source          1..8533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..57
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     42..58
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        86..750
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    302..419
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     714..737
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(2133..2135,2136..2138,2139..2849)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2133..2135,2136..2138,2139..2849)
                     /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(2178..2199)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2439..2458)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2786..2807
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2862..2891
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4566..4574
                     /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(5039..5382)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      complement(6738..7326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6818..6837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     tRNA            complement(join(7369..7388,7389..7473,7474..7548))
                     /product="E. coli amber-suppressor tyrosine tRNA"
                     /label=supF
     primer_bind     complement(7653..7672)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7664..8479
                     /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             7664..8479
                     /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     complement(7737..7756)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gacggcgccg gatccgtcga ggtaccgggc cccccctcga tcgaggtcga cggtatcgat
       61 aagcttgata tcgaattcct tgaattttca aaaattctta cttttttttt ggatggacgc
      121 aaagaagttt aataatcata ttacatggca ttaccaccat atacatatcc atatacatat
      181 ccatatctaa tcttacttat atgttgtgga aatgtaaaga gccccattat cttagcctaa
      241 aaaaaccttc tctttggaac tttcagtaat acgcttaact gctcattgct atattgaagt
      301 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt
      361 cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga
      421 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac
      481 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga
      541 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat
      601 taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc
      661 ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac
      721 ctctatactt taacgtcaag gagaaaaaac cccggatcat gaaggttttg attgtcttgt
      781 tggctatctt cgctgctttg cccttggctc tagctcaacc ggttatttct actaccgtcg
      841 gttccgctgc agaaggctct ttggacaaga gagaagctcg gccggacgtc atgcccatca
      901 tgggctcctc ggtgtacatc acggtggagc tggccattgc tgtgctggcc atcctgggca
      961 atgtgctggt gtgctgggcc gtgtggctca acagcaacct gcagaacgtc accaactact
     1021 ttgtggtgtc actggcggcg gccgacatcg cagtgggtgt gctcgccatc ccctttgcca
     1081 tcaccatcag caccgggttc tgcgctgcct gccacggctg cctcttcatt gcctgcttcg
     1141 tcctggtcct cacgcagagc tccatcttca gtctcctggc catcgccatt gaccgctaca
     1201 ttgccatccg catcccgctc cggtacaatg gcttggtgac cggcacgagg gctaagggca
     1261 tcattgccat ctgctgggtg ctgtcgtttg ccatcggcct gactcccatg ctaggttgga
     1321 acaactgcgg tcagccaaag gagggcaaga accactccca gggctgcggg gagggccaag
     1381 tggcctgtct ctttgaggat gtggtcccca tgaactacat ggtgtacttc aacttctttg
     1441 cctgtgtgct ggtgcccctg ctgctcatgc tgggtgtcta tttgcggatc ttcctggcgg
     1501 cgcgacgaca gctgaagcag atggagagcc agcctctgcc gggggagcgg gcacggtcca
     1561 cactgcagaa ggaggtccat gctgccaagt cactggccat cattgtgggg ctctttgccc
     1621 tctgctggct gcccctacac atcatcaact gcttcacttt cttctgcccc gactgcagcc
     1681 acgcccctct ctggctcatg tacctggcca tcgtcctctc ccacaccaat tcggttgtga
     1741 atcccttcat ctacgcctac cgtatccgcg agttccgcca gaccttccgc aagatcattc
     1801 gcagccacgt cctgaggcag caagaacctt tcaaggcagc tggcaccagt gcccgggtct
     1861 tggcagctca tggcagtgac ggagagcagg tcagcctccg tctcaacggc cacccgccag
     1921 gagtgtgggc caacggcagt gctccccacc ctgagcggag gcccaatggc tacgccctgg
     1981 ggctggtgag tggagggagt gcccaagagt cccaggggaa cacgggcctc ccagacgtgg
     2041 agctccttag ccatgagctc aagggagtgt gcccagagcc ccctggccta gatgaccccc
     2101 tggcccagga tggagcagga gtgtccccgc ggatggtgag caagggcgag gagctgttca
     2161 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2221 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     2281 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     2341 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2401 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2461 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2521 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2581 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2641 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2701 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2761 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2821 tcactctcgg catggacgag ctgtacaagc acgtgtcatc acatcatcat catcatcatc
     2881 atcatcatca ttaataactt aagagttgaa gacatgggta tctattactg tacgggttct
     2941 tactatggta tggactactg gggtcaagga acctcagtca ccgtctcctg ataacaaaag
     3001 cttatttctg aagaagactt gtaatagctc gagatctgat aacaacagtg tagatgtaac
     3061 aaaatcgact ttgttcccac tgtactttta gctcgtacaa aatacaatat acttttcatt
     3121 tctccgtaaa caacatgttt tcccatgtaa tatccttttc tatttttcgt tccgttacca
     3181 actttacaca tactttatat agctattcac ttctatacac taaaaaacta agacaatttt
     3241 aattttgctg cctgccatat ttcaatttgt tataaattcc tataatttat cctattagta
     3301 gctaaaaaaa gatgaatgtg aatcgaatcc taagagaatt gagctctgca gcatgctcga
     3361 cctgcggatc taatgaatcc atttgttagt taatagttta aatgttttta tcggaagagg
     3421 ttttgtcatc acatcagcaa tgttcttatt gactcctcgt caattctctt aggattcgat
     3481 tcacattcat ctttttttag ctactaatag gataaattat aggaatttat aacaaattga
     3541 aatatggcag gcagcaaaat taaaattgtc ttagtttttt agtgtataga agtgaatagc
     3601 tatataaagt atgtgtaaag ttggtaacgg aacgaaaaat agaaaaggat attacatggg
     3661 aaaacatgtt gtttacggag aaatgaaaag tatattgtat tttgtacgag ctaaaagtac
     3721 agtgggaaca aagtcgattt tgttacatct acactgttgt tatcagatct cgagctatta
     3781 caagtcttct tcagaaataa gcttttgtta tcaggagacg gtgactgagg ttccttgacc
     3841 ccagtagtcc ataccatagt aagaacccgt acagtaatag atacccatgt cttcaactct
     3901 taagctaaca aatcgggttg gtcaatcctc tagtccaagc ataggatccg agagttatca
     3961 ccaataagca atatgaaatt gcaatcgaaa cagtataata aataagcatg ttaataaatg
     4021 atagcttata cccttcattg ataaacgtgc taatggtact caaagtgccg caaaatccgg
     4081 atattaacgc agaaacaata tgacatgaat tcaacgagtt cacaattgga acgtccgtag
     4141 aaaagtgttt tttaccacgt tgcaccatgg taaatatgcc aatcaataaa gtggcgaaaa
     4201 cgtttgccaa gaaagtaccc aatggaaact ttttgtttgt tttattaaac atttctgcca
     4261 accaatatct taggaaacca gcgaaaattc caaatagaca tggtagtgtc cacttgcccc
     4321 tcgaatagtt ctcataatac gcacacagca ccacaaacaa aataattaat ggaaaagcta
     4381 gtgcatacgc gagtttatcc acaatatcaa aaaatttctt aaaaaatggt gctggagttt
     4441 tgaatttaaa ctctagaata tttttttcca tttcttgtgt atcaacatca ccagcatttt
     4501 cctttacagt gtctgaggga ggggtatatg gcttcgaaaa cgaactagat ccataagcaa
     4561 caataacctc cttaccaagt tgcctaccga aaataagact acccatggaa accataagat
     4621 gaacgagcaa gacagataaa aactccatta taccgtaagc tctgttcggc agttttgtgt
     4681 ggttagcgat attaccgtta gttaagtttg tagagtgttc gaacatctct agcagcatac
     4741 tagaaaagga agacagggca ccacagtacc cagtagttac acctaaaaac aagacttggt
     4801 gatctttcat ccaagtgtaa gcatttaaag attgcataat ccccattagc atgcaggatg
     4861 aacaattaga ccagagaacg gtcggagcag taacgaaggc cggagtgtag gtggaaagcg
     4921 ccgttatagc ctgacgtgtt tcagtaccta atatgcagaa cgtggtgaat gtacaaaaaa
     4981 catgaatata atggctcaac ttgtgatttg atatgaccgg attgaaaatc atcggccggg
     5041 ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa aaggaagtat
     5101 atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca ggggcgcggc
     5161 gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg tctgcagcga
     5221 ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta
     5281 tacatgggga tgtatgggct aaatgtacgg gcgacagtca catcatgccc ctgagctgcg
     5341 cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctggccg ggtgacccgg
     5401 cggggacgag gcaagctaaa cagatctagg tcagctcgta ggtatttgaa gtcaccggat
     5461 agacctttaa gtattagttg acaggccaat ctgtcgctaa cattgatatt gttttctttt
     5521 agcctctgaa tgatcgtact gactgtaatt tcaaatgtat cagcagatgt acttccgtcg
     5581 tactcaaggt tggcaagagc tatccaatcc tttaattctt gattgttagt ttgcattttg
     5641 gacacacttt tacaaaggac tgtaaggatg tcagcataat taatttctaa aatttgtttt
     5701 acccatgttg gcaataaatg aaatggggca aatgcttgga aggtattgta tatatacgca
     5761 tgttcttcat aagtcatttg tcttttgatt tcaccctggt catttggaat gatgtcaccg
     5821 agattagagt tcttcaaaaa tctgatgtaa aatttaaccc atgtagaaaa gttttcttct
     5881 gatgttaaag tgtgtggtgg taagacgtta tttcccacct ttgtcttagc ttgaggtatt
     5941 tcagagtgtt gattttttaa atcgattgga tcaggtgagg aagtactcag tggtgggata
     6001 tactgtggca acggatagtg tgggtccggt tgataatacg caggtgacgt ttgataatgt
     6061 gaacacgtca tcatagatgg ttgttggtaa tgtgcccagt taggggccat agctttgttt
     6121 ggggtcatca tgccgtgctg ttggtactgt ccattctgtg gaggtggtac tgaagcaggt
     6181 tgaggagaga catgatgatg gttctctgga acagctgatg tcccaggtgt tgtctcttgt
     6241 tgagaattaa ccttagtgga atctctatca aattccggta aattggaagc tgaaacggct
     6301 aacggatctt gatttgattg gacttcctta gaagtaaccg aagcataggc gctaccatga
     6361 agattgggtg aattttgaga taattgttgg gattccattt ttaataaggc aataatatta
     6421 ggtatgtaga tatactagaa gttctcctcg aggatttagg aatccataaa agggaatctg
     6481 caattctaca caattctata aatattatta tcatcatttt atatgttaat attcattgat
     6541 cctattacat tatcaatcct tgcgtttcag cttccactaa tttagatgac tatctgtcat
     6601 caattgcgtc atcttctaac accgtatatg ataatatact agtaacgtaa atactagtta
     6661 gtagatgata gttgattttt attccaacat accacccata atgtaataga tctctagaag
     6721 cttgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6781 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6841 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6901 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6961 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7021 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7081 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7141 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     7201 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7261 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7321 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcaattctt tcggactttt
     7381 gaaagtgatg gtgggggggg aaggattcga accttcgaag tcgatgacgg cagatttaga
     7441 gtctgctccc tttggccgct cgggaacccc accacaggta atgcttttac tggcctgctc
     7501 ccttatcggg aagcggggcg catcatatca aatgacgcgc cgctgtaaag tgttacgttg
     7561 agaaagaatt accggggatc cgggttacat tgcacaagat aaaaatatat catcatgaac
     7621 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     7681 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     7741 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     7801 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     7861 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     7921 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     7981 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     8041 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     8101 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     8161 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt
     8221 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     8281 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     8341 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     8401 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     8461 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     8521 gctgacttga cgg
//
