LOCUS       sequence-403573-        6668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses POWV NS2B-NS3 protease (with GS linker) from a GAL10 
            promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6668)
  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 6668)
  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-POWV_NS2B-GS-NS3
FEATURES             Location/Qualifiers
     source          1..6668
                     /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(2656..2672)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2657..2673)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2675..2922
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2691..2709)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2940..2959)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2941..2959)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2969..2986)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2969..2985)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2978..3000)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3126..3581
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3213..3232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3423..3444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3712..4515)
                     /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(4516..4731)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4755..4774)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4874..4896
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4934..4952)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4993..5496
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5533..5637
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5638..5706,5707..6498)
                     /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(5856..5875)
                     /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 cccgtagact ccagctaacg gcggaatggg ccggagtggt ggagtggaac cctgagttgg
     1921 tcaatgaggg aggtgaggta agcttgaagg tcagacagga tgccatgggg aacttgcact
     1981 tgacagaggt ggaacgggag gagcgtcgtt tggccggagg tggaggttca ggggggggtg
     2041 gaactgacct ggttttttca gggcagttgc ctgaccaagg ggaaaaacgt tcctttgaca
     2101 tcaaggaagg cgtataccgc atatacgccc ctgggctctt ctggggttac cgccagatag
     2161 gtgtgggata tggaacgaaa ggagtcttac acaccatgtg gcatgtgaca agaggagccg
     2221 ccctcagcgt ggagggggcc acatcaggac catactgggc ggatgtgcgt gaagacgtgg
     2281 tctgctatgg gggggcctgg ggcttggaca aaaaatgggg aggcgaagtc gttcaggtac
     2341 acgcattccc tccggacagt ggacacaaaa tccatcaatg ccagccagga aagctgaacc
     2401 ttgagggggg gagagttctg ggggccatcc cgatagacct cccacgtggt acttcaggta
     2461 gcccaatcat caatgcacag ggagatgtgc tgggtctgta cggcaatggt ctgaaatcaa
     2521 atgatgttta catcagtagc attgctcagg gcaatgttga aaagagtcgc cctgagatgc
     2581 cattggcagt ccagtaaaac ccagctttct tgtactaagt ggtgatgggc tgcaggaatt
     2641 cgatatcaag cttatcgata ccgtcgacct cgagtcatgt aattagttat gtcacgctta
     2701 cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa
     2761 gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt
     2821 caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc
     2881 ttgcttgaga aggttttggg acgctcgaag gctttaattt gcggccggta cccaattcgc
     2941 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3001 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3061 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3121 aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3181 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3241 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3301 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3361 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3421 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3481 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3541 atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc
     3601 cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata attaaattga
     3661 agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg
     3721 ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac
     3781 cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt
     3841 agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa
     3901 acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct
     3961 ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt
     4021 agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc
     4081 tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc
     4141 caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga
     4201 gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa
     4261 attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt
     4321 caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc
     4381 cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg
     4441 catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata
     4501 tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt
     4561 taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct
     4621 aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt
     4681 tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta
     4741 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4801 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4861 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4921 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4981 gtttcttaga cggatcgctt gcctgtaact tacacgcgcc tcgtatcttt taatgatgga
     5041 ataatttggg aatttactct gtgtttattt atttttatgt tttgtatttg gattttagaa
     5101 agtaaataaa gaaggtagaa gagttacgga atgaagaaaa aaaaataaac aaaggtttaa
     5161 aaaatttcaa caaaaagcgt actttacata tatatttatt agacaagaaa agcagattaa
     5221 atagatatac attcgattaa cgataagtaa aatgtaaaat cacaggattt tcgtgtgtgg
     5281 tcttctacac agacaagatg aaacaattcg gcattaatac ctgagagcag gaagagcaag
     5341 ataaaaggta gtatttgttg gcgatccccc tagagtcttt tacatcttcg gaaaacaaaa
     5401 actatttttt ctttaatttc tttttttact ttctattttt aatttatata tttatattaa
     5461 aaaatttaaa ttataattat ttttatagca cgtgatgaaa aggacccagg tggcactttt
     5521 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5581 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5641 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5701 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5761 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5821 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5881 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5941 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6001 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6061 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6121 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6181 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6241 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6301 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6361 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6421 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6481 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6541 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6601 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6661 ggatcttc
//
