LOCUS       sequence-402848-        7192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HCV NS3-NS4A protease (with GS linker) from a GAL promoter
            with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7192)
  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 7192)
  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: pAG426GAL-HCV_NS3-GS-NS4A
FEATURES             Location/Qualifiers
     source          1..7192
                     /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"
     protein_bind    complement(2294..2318)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2351..2367)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2352..2368)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2370..2617
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2386..2404)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2635..2654)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2636..2654)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2664..2681)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2664..2680)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2673..2695)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2821..3276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2908..2927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3118..3139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3407..4210)
                     /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(4211..4426)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4450..4469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4569..4591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4629..4647)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4688..6030
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5020..5067
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        6057..6161
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6162..6230,6231..7022)
                     /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(6380..6399)
                     /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 aaaatggcac caattactgc ctatgctcaa caaacccgtg gtcttttagg atgcattatt
     1621 acatcattaa caggaaggga taagaatcag gttgaagggg aagtgcaaat tgtctctaca
     1681 gcgactcaga ccttcttggc tacatgtatt aacggagtgt gttggacagt ttatcatggt
     1741 gctggcacca gaacaatagc tagtcctaaa gggccagtaa ttcaaatgta tactaacgta
     1801 gatcaggatc tagttggttg gccagcacca cagggcagta ggagcttaac tccatgtact
     1861 tgtggtagtt ctgatttgta tttagttact cgtcatgctg acgttatccc agttaggaga
     1921 agaggagact ccagaggctc attgctaagt ccaaggccga tatcttatct gaaggggagc
     1981 tcaggtgggc cattgctgtg tccggctggc catgcagttg gtttatttag agcagctgtt
     2041 tgtacgagag gtgttgcaaa agctgtcgat ttcattccag ttgaaaatct ggaaacgacg
     2101 atgaggagcc cagtatttac agataattca agcccacctg cggtaacatt aaccggagga
     2161 ggtggcagtg gtggtggtgg ttctggcggc ggtggatctg gtggcggtgg atccggatgt
     2221 gttgtaattg tcggtagaat agttttatct ggcaaaccag ccataatccc agatagagaa
     2281 gtcttatatt aaaacccagc tttcttgtac aaagtggtga tgggctgcag gaattcgata
     2341 tcaagcttat cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc
     2401 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     2461 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     2521 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     2581 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat
     2641 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2701 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2761 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2821 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2881 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2941 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3001 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3061 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3121 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3181 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3241 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     3301 gcatctgtgc ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc
     3361 taatttgtga gtttagtata catgcattta cttataatac agttttttag ttttgctggc
     3421 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag
     3481 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga
     3541 ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca
     3601 caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag
     3661 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat
     3721 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag
     3781 gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca
     3841 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact
     3901 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt
     3961 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga
     4021 tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta
     4081 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga
     4141 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag
     4201 ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga
     4261 atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc
     4321 tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaag
     4381 gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg tggtatggtg
     4441 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4501 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4561 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4621 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4681 ttagtatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga
     4741 ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc
     4801 atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg
     4861 catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat
     4921 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg
     4981 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc
     5041 ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag
     5101 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
     5161 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
     5221 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
     5281 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
     5341 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
     5401 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
     5461 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
     5521 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
     5581 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
     5641 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
     5701 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
     5761 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
     5821 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
     5881 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
     5941 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
     6001 ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg
     6061 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     6121 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     6181 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6241 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     6301 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     6361 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     6421 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     6481 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6541 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6601 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6661 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6721 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6781 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6841 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6901 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6961 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     7021 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     7081 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7141 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
