LOCUS       sequence-403575-        6255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EMCV 3C protease from a GAL promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6255)
  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 6255)
  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-EMCV_3C
FEATURES             Location/Qualifiers
     source          1..6255
                     /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"
     primer_bind     complement(2243..2259)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2244..2260)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2262..2509
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2278..2296)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2527..2546)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2528..2546)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2556..2573)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2556..2572)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2565..2587)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2713..3168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2800..2819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3010..3031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3299..4102)
                     /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(4103..4318)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4342..4361)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4461..4483
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4521..4539)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4580..5083
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5120..5224
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5225..5293,5294..6085)
                     /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(5443..5462)
                     /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 aaaatgggac caaaccctgt gatggacttt gaaaaatatg tagccaaaca tgtaaccgcc
     1621 cccattggtt ttgtctaccc cactggggtg agcacccaga cttgcctcct tgtgagaggc
     1681 cgcaccttgg tagtaaatag acacatggcc gagtctgact ggacttccat agtagtgcgt
     1741 ggagtcacac acgcccgctc tactgttaaa attttggcca tagctaaagc aggcaaagag
     1801 actgacgtat ctttcatccg cctctcttct ggtcctctat tcagagacaa tacatccaaa
     1861 tttgtcaagg ctggtgatgt acttcctact ggtgccgctc cagtcacggg gataatgaac
     1921 acggacatac ccatgatgta cacaggaacc ttcctgaaag ctggtgtgtc agtcccagtg
     1981 gaaaccggcc agacctttaa tcactgtatt cattacaagg ctaacacacg aaagggctgg
     2041 tgtggatcag ccctactggc agatcttgga ggaagcaaga aaatccttgg catccattct
     2101 gctggctcta tgggaatagc cgccgcctcg attgtgtcac aggagatgat tcgggcggta
     2161 gtgaatgcct ttgagccaca gtaaaaccca gctttcttgt actaagtggt gatgggctgc
     2221 aggaattcga tatcaagctt atcgataccg tcgacctcga gtcatgtaat tagttatgtc
     2281 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca
     2341 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt
     2401 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact
     2461 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc
     2521 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     2581 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2641 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2701 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2761 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2821 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2881 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2941 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3001 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3061 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3121 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
     3181 ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt
     3241 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt
     3301 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac
     3361 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag
     3421 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt
     3481 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca
     3541 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag
     3601 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca
     3661 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac
     3721 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac
     3781 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga
     3841 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa
     3901 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa
     3961 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct
     4021 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt
     4081 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca
     4141 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata
     4201 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa
     4261 aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa
     4321 ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4381 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4441 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4501 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4561 aataatggtt tcttagacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa
     4621 tgatggaata atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat
     4681 tttagaaagt aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa
     4741 ggtttaaaaa atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc
     4801 agattaaata gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg
     4861 tgtgtggtct tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa
     4921 gagcaagata aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa
     4981 aacaaaaact attttttctt taatttcttt ttttactttc tatttttaat ttatatattt
     5041 atattaaaaa atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg
     5101 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5161 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5221 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5281 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5341 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5401 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5461 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5521 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5581 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5641 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5701 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5761 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5821 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5881 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5941 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6001 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6061 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6121 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6181 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6241 gatcaaagga tcttc
//
