LOCUS       sequence-402619-        6533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HPeV 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 6533)
  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 6533)
  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-HPeV_3C
FEATURES             Location/Qualifiers
     source          1..6533
                     /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(2521..2537)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2522..2538)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2540..2787
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2556..2574)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2805..2824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2806..2824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2834..2851)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2834..2850)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2843..2865)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2991..3446
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3078..3097)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3288..3309
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3577..4380)
                     /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(4381..4596)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4620..4639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4739..4761
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4799..4817)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4858..5361
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5398..5502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5503..5571,5572..6363)
                     /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(5721..5740)
                     /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 tacaaaatga
     1861 gggagttcaa gaatgaagcg ccatatgatg gccaactgga gcatatcatc tcccagatgg
     1921 catacatcac tggctcaacc acaggccaca taacccattg tgcaggttac cagcatgatg
     1981 agatcatact gcatggacac tcaatcaaat atttagaaca agaagaagaa ctgacattac
     2041 attataagaa taaagttttc ccaattgagc aaccatctgt cactcaggta acgcttggtg
     2101 gtaaacctat ggatttggcc attgtaaaat gtaaattgcc attcagattc aagaaaaact
     2161 ctaaatatta tactaataaa attgggactg agagcatgtt gatctggatg actgagcagg
     2221 gtatcatcac aaaagaagtt cagagggtgc atcattctgg tggcattaaa actagagagg
     2281 ggactgaaag cactaagacc atcagctaca cggtgaaatc ttgcaaaggt atgtgtggtg
     2341 gacttttgat ctcaaaagtg gaaggcaatt ttaagattct tggtatgcac attgctggga
     2401 atggtgagat gggtgtagct atccccttta attttcttaa aaatgacatg tctgatcagt
     2461 aaaacccagc tttcttgtac taagtggtga tgggctgcag gaattcgata tcaagcttat
     2521 cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc
     2581 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
     2641 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     2701 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
     2761 ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt
     2821 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2881 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2941 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     3001 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3061 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3121 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3181 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3241 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3301 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3361 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3421 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     3481 ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc taatttgtga
     3541 gtttagtata catgcattta cttataatac agttttttag ttttgctggc cgcatcttct
     3601 caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc
     3661 ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc
     3721 cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt
     3781 cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc
     3841 gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt
     3901 agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt
     3961 tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc
     4021 attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac
     4081 tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga
     4141 taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg
     4201 tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt
     4261 ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag
     4321 cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat
     4381 gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca
     4441 atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt
     4501 ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa
     4561 tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt
     4621 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4681 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4741 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4801 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacggat
     4861 cgcttgcctg taacttacac gcgcctcgta tcttttaatg atggaataat ttgggaattt
     4921 actctgtgtt tatttatttt tatgttttgt atttggattt tagaaagtaa ataaagaagg
     4981 tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat ttcaacaaaa
     5041 agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga tatacattcg
     5101 attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc tacacagaca
     5161 agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa aggtagtatt
     5221 tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat tttttcttta
     5281 atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat ttaaattata
     5341 attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg aaatgtgcgc
     5401 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5461 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5521 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5581 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5641 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5701 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5761 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5821 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5881 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5941 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6001 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6061 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6121 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6181 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6241 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6301 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6361 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6421 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6481 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
