LOCUS       sequence-402850-        6476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NWV 3CL protease from a GAL10 promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6476)
  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 6476)
  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: pAG416GAL10-NWV_3CL
FEATURES             Location/Qualifiers
     source          1..6476
                     /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        complement(1108..1549)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(1114..1137)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1432..1549
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    1827..1851
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2464..2480)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2465..2481)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2483..2730
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2499..2517)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2748..2767)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2749..2767)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2777..2794)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2777..2793)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2786..2808)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2934..3389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3021..3040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3231..3252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3520..4323)
                     /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(4324..4539)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4563..4582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4682..4704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4742..4760)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4801..5304
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5341..5445
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5446..5514,5515..6306)
                     /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(5664..5683)
                     /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 tgaccatgat tacgccaagc ggggtgtgtt
     1081 gttggtgggt tgggtgtata gttttttctc cttgacgtta aagtatagag gtatattaac
     1141 aattttttgt tgatactttt atgacatttg aataagaagt aatacaaact gaaaatgttg
     1201 aaagtattag ttaaagtggt tatgcagctt ttccatttat atatctgtta atagatcaaa
     1261 aatcatcgct tcgctgatta attaccccag aaataaggct aaaaaactaa tcgcattatc
     1321 atcctatggt tgttaatttg attcgttaat ttgaaggttt gtggggccag gttactgcca
     1381 atttttcctc ttcataacca taaaagctag tattgtagaa tctttattgt tcggagcagt
     1441 gcggcgcgag gcacatctgc gtttcaggaa cgcgaccggt gaagacgagg acgcacggag
     1501 gagagtcttc cgtcggaggg ctgtcgcccg ctcggcggct tctaatccgt acttcaatat
     1561 agcaatgagc agttaagcgt attactgaaa gttccaaaga gaaggttttt ttaggctaag
     1621 ataatggggc tctttacatt tccacaacat ataagtaaga ttagatatgg atatgtatat
     1681 ggtggtaatg ccatgtaata tgattattaa acttctttgc gtccatccaa aaaaaaagta
     1741 agaatttttg aaaattcaat ataaatgtct aaaggtgaag aattattcag aattccagct
     1801 gaccaccgac tagtggatcc cccatcacaa gtttgtacaa aaaagcaggc tacaaaatgg
     1861 ctcccccgac actatggagc cgagtcacaa agtttggatc aggatggggc ttttgggtca
     1921 gcccgacagt gttcatcaca accacacatg tagtgccaac tggtgtgaaa gaattctttg
     1981 gtgagcccct atctagtata gcaatccacc aagcaggtga gttcacacaa ttcaggttct
     2041 caaagaaaat gcgccctgac ttgacaggta tggtccttga agaaggttgc cctgaaggga
     2101 cagtctgctc agtcctaatt aaacgggatt cgggtgaact acttccgcta gccgtccgta
     2161 tgggggctat tgcctccatg aggatacagg gtcggcttgt ccatggccaa tcagggatgt
     2221 tactgacagg ggccaatgca aaggggatgg atcttggcac tataccagga gactgcgggg
     2281 caccatacgt ccacaagcgc gggaatgact gggttgtgtg tggagtccac gctgcagcca
     2341 caaagtcagg caacaccgtg gtctgcgctg tacaggctgg agagggcgaa accgcactag
     2401 aataaaaccc agctttcttg tactaagtgg tgatgggctg caggaattcg atatcaagct
     2461 tatcgatacc gtcgacctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct
     2521 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     2581 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     2641 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     2701 gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt
     2761 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     2821 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     2881 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc
     2941 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3001 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3061 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3121 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3181 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3241 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3301 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3361 ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg
     3421 tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag ctctaatttg
     3481 tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct
     3541 tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct
     3601 tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc
     3661 atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg
     3721 tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa
     3781 gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc
     3841 agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt
     3901 tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg
     3961 tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt
     4021 gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc
     4081 ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac
     4141 atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt
     4201 ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa
     4261 tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga
     4321 catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta agaatactgg
     4381 gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc
     4441 cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg
     4501 aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg gtgcactctc
     4561 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4621 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4681 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4741 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4801 gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat aatttgggaa
     4861 tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag taaataaaga
     4921 aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa aatttcaaca
     4981 aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat agatatacat
     5041 tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc ttctacacag
     5101 acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat aaaaggtagt
     5161 atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac tattttttct
     5221 ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa aatttaaatt
     5281 ataattattt ttatagcacg tgatgaaaag gacccaggtg gcacttttcg gggaaatgtg
     5341 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5401 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5461 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5521 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5581 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5641 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5701 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5761 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5821 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5881 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5941 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6001 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6061 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6121 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6181 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6241 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6301 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6361 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6421 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
