LOCUS       sequence-402623-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EYFP from a GAL promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Hong SJ, Resnick SJ, Iketani S, Cha JW, Albert BA, Fazekas CT, Chang
            CW, Liu H, Dagan S, Abagyan MR, Fajtova P, Culbertson B, Brace B, 
            Reddem ER, Forouhar F, Glickman JF, Balkovec JM, Stockwell BR, 
            Shapiro L, O'Donoghue AJ, Sabo Y, Freundlich JS, Ho DD, Chavez A
  TITLE     A multiplex method for rapidly identifying viral protease 
            inhibitors.
  JOURNAL   Mol Syst Biol. 2025 Feb;21(2):158-172. doi: 
            10.1038/s44320-024-00082-1. Epub 2025 Jan 6.
  PUBMED    39762652
REFERENCE   2  (bases 1 to 6354)
  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: 
            "10.1038/s44320-024-00082-1"; journalName: "Mol Syst Biol"; date: 
            "2025-02"; volume: "21"; issue: "2"; pages: "158-172"
COMMENT     Sequence Label: pAG416GAL-EYFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1054..1495
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    1054..1171
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1466..1489
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1510..1526
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1510..1526
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1534..1558
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1564..1566,1567..1569,1570..2283)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1609..1630)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1870..1889)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2217..2238
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2285..2309)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2342..2358)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2343..2359)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2361..2608
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2377..2395)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2626..2645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2627..2645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2655..2672)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2655..2671)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2664..2686)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2812..3267
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2899..2918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3109..3130
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3398..4201)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4202..4417)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4441..4460)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4560..4582
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4620..4638)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4679..5182
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5219..5323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5324..5392,5393..6184)
                     /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(5542..5561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctcta gtacggatta gaagccgccg agcgggtgac
     1081 agccctccga aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa
     1141 acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt
     1201 tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga
     1261 atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta
     1321 attaatcagc gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca
     1381 taaccacttt aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta
     1441 ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa
     1501 accccggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa agcaggctac
     1561 aaaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1621 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1681 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1741 accctcgtga ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg
     1801 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1861 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1921 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1981 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2041 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2101 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2161 cactacctga gctaccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     2221 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2281 taaaacccag ctttcttgta caaagtggtg atgggctgca ggaattcgat atcaagctta
     2341 tcgataccgt cgacctcgag tcatgtaatt agttatgtca cgcttacatt cacgccctcc
     2401 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat
     2461 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt
     2521 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt
     2581 tttgggacgc tcgaaggctt taatttgcgg ccggtaccca attcgcccta tagtgagtcg
     2641 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2701 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2761 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct
     2821 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2881 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2941 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3001 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3061 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3121 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3181 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3241 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     3301 cggtatttca caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg
     3361 agtttagtat acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc
     3421 tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc
     3481 cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag accacatcat
     3541 ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg
     3601 tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc
     3661 cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag
     3721 tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg
     3781 ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg
     3841 cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga
     3901 ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg
     3961 ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat
     4021 gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg
     4081 tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata
     4141 gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca
     4201 tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc
     4261 aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct
     4321 tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa
     4381 atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt gcactctcag
     4441 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4501 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4561 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4621 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgga
     4681 tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa tttgggaatt
     4741 tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta aataaagaag
     4801 gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa
     4861 aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag atatacattc
     4921 gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac
     4981 aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat
     5041 ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta ttttttcttt
     5101 aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa tttaaattat
     5161 aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg gaaatgtgcg
     5221 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5281 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5341 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5401 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5461 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5521 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5581 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5641 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5701 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5761 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5821 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5881 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5941 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6001 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6061 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6121 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6181 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6241 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6301 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
