LOCUS       sequence-402803-        6482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PV 3C 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 6482)
  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 6482)
  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-PV_3C
FEATURES             Location/Qualifiers
     source          1..6482
                     /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(2470..2486)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2471..2487)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2489..2736
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2505..2523)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2754..2773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2755..2773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2783..2800)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2783..2799)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2792..2814)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2940..3395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3027..3046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3237..3258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3526..4329)
                     /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(4330..4545)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4569..4588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4688..4710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4748..4766)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4807..5310
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5347..5451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5452..5520,5521..6312)
                     /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(5670..5689)
                     /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 gaccagggtt cgattacgca gtggctatgg ctaaaagaaa cattgttaca gcaactacta
     1921 gcaagggaga gttcactatg ttaggagtcc acgacaacgt ggctatttta ccaacccacg
     1981 cttcacctgg tgaaagcatt gtgatcgatg gcaaagaagt ggagatcttg gatgccaaag
     2041 cgctcgaaga tcaagcagga accaatcttg aaatcactat aatcactcta aagagaaatg
     2101 aaaagttcag agacattaga ccacatatac ctactcaaat cactgagaca aatgatggag
     2161 tcttgatcgt gaacactagc aagtacccca atatgtatgt tcctgtcggt gctgtgactg
     2221 aacagggata tctaaatctc ggtgggcgcc aaactgctcg tactctaatg tacaactttc
     2281 caaccagagc aggacagtgt ggtggagtca tcacatgtac tgggaaagtc atcgggatgc
     2341 atgttggtgg gaacggttca cacgggtttg cagcggccct gaagcgatca tacttcactc
     2401 agagtcaata aaacccagct ttcttgtact aagtggtgat gggctgcagg aattcgatat
     2461 caagcttatc gataccgtcg acctcgagtc atgtaattag ttatgtcacg cttacattca
     2521 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     2581 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     2641 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     2701 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat tcgccctata
     2761 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2821 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2881 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2941 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3001 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3061 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3121 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3181 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3241 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3301 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3361 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     3421 catctgtgcg gtatttcaca ccgcataggg taataactga tataattaaa ttgaagctct
     3481 aatttgtgag tttagtatac atgcatttac ttataataca gttttttagt tttgctggcc
     3541 gcatcttctc aaatatgctt cccagcctgc ttttctgtaa cgttcaccct ctaccttagc
     3601 atcccttccc tttgcaaata gtcctcttcc aacaataata atgtcagatc ctgtagagac
     3661 cacatcatcc acggttctat actgttgacc caatgcgtct cccttgtcat ctaaacccac
     3721 accgggtgtc ataatcaacc aatcgtaacc ttcatctctt ccacccatgt ctctttgagc
     3781 aataaagccg ataacaaaat ctttgtcgct cttcgcaatg tcaacagtac ccttagtata
     3841 ttctccagta gatagggagc ccttgcatga caattctgct aacatcaaaa ggcctctagg
     3901 ttcctttgtt acttcttctg ccgcctgctt caaaccgcta acaatacctg ggcccaccac
     3961 accgtgtgca ttcgtaatgt ctgcccattc tgctattctg tatacacccg cagagtactg
     4021 caatttgact gtattaccaa tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta
     4081 cttggcggat aatgccttta gcggcttaac tgtgccctcc atggaaaaat cagtcaagat
     4141 atccacatgt gtttttagta aacaaatttt gggacctaat gcttcaacta actccagtaa
     4201 ttccttggtg gtacgaacat ccaatgaagc acacaagttt gtttgctttt cgtgcatgat
     4261 attaaatagc ttggcagcaa caggactagg atgagtagca gcacgttcct tatatgtagc
     4321 tttcgacatg atttatcttc gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa
     4381 tactgggcaa tttcatgttt cttcaacact acatatgcgt atatatacca atctaagtct
     4441 gtgctccttc cttcgttctt ccttctgttc ggagattacc gaatcaaaaa aatttcaagg
     4501 aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc
     4561 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4621 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4681 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4741 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4801 tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga tggaataatt
     4861 tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt agaaagtaaa
     4921 taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt
     4981 tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga ttaaatagat
     5041 atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt gtggtcttct
     5101 acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagag caagataaaa
     5161 ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac aaaaactatt
     5221 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
     5281 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
     5341 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5401 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6001 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6061 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6121 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6181 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6301 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6361 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6421 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6481 tc
//
