LOCUS       sequence-402629-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KUNV NS2B-NS3 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 7204)
  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 7204)
  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-KUNV_NS2B-GS-NS3
FEATURES             Location/Qualifiers
     source          1..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..78
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    195..216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(231..236,237..254,255..261)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    269..285
                     /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     274..296
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     293..309
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     293..309
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     328..348
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        330..348
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        372..813
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    372..489
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     784..807
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     828..844
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     828..844
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    852..876
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1624..1648)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1681..1697)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1682..1698)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1700..1947
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1716..1734)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1965..1984)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1966..1984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1994..2011)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1994..2010)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2003..2025)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2151..2606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2238..2257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2448..2469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2737..3540)
                     /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(3541..3756)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3780..3799)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3899..3921
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3959..3977)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4018..5360
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4350..4397
                     /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        5387..5491
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5492..5560,5561..6352)
                     /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(5710..5729)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6523..7111
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7012..7031
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
       61 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
      121 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      181 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
      241 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
      301 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
      361 gagctctagt acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc
      421 ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc
      481 actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat
      541 tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat
      601 gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt
      661 tttgatctat taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt
      721 caacattttc ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt
      781 gttaatatac ctctatactt taacgtcaag gagaaaaaac cccggattct agaactagtg
      841 gatcccccat cacaagtttg tacaaaaaag caggctacaa aatgtctgga aaatcgaccg
      901 acatgtggat tgagaggaca gcggacatct cctgggaggg tgatgcggaa atcacaggtt
      961 ccagtgaaag agttgatgtt cggcttgacg atgacgggaa cttccagctc atgaatgatc
     1021 caggagcacc ttggaaaata ggaggtggag gttcaggggg gggtggagga ggtgtgctgt
     1081 gggacactcc ctctccaaag gagtacaaga gaggggacac gaccactggt gtctatagga
     1141 tcatgactcg tggattactt ggtagttacc aagcaggagc aggcgtgatg gttgaaggtg
     1201 ttttccacac cctctggcac acgacaaaag gagccgctct gatgagcgga gaaggccgcc
     1261 tggatccata ttggggcagc gtcaaggagg atcgactttg ttatggtgga ccctggaaac
     1321 tgcagcacaa gtggaatggg caagatgagg tgcaaatgat tgtggtggaa ccagggaaga
     1381 atgtcaagaa cgtccagact aagccggggg tgttcaaaac acctgaaggg gagatcgggg
     1441 ccgtgactct ggatttcccc actggaacat caggctcgcc catagtggac aaaaacggtg
     1501 atgtgattgg gctttatggc aacggagtca taatgcccaa tggctcatac ataagcgcga
     1561 tagtgcaagg tgaaaggatg gatgagccgg ttcccgccgg attcgaacct gagatgttgt
     1621 aaaacccagc tttcttgtac aaagtggtga tgggctgcag gaattcgata tcaagcttat
     1681 cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc
     1741 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
     1801 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     1861 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
     1921 ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt
     1981 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2041 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2101 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     2161 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2221 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2281 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2341 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2401 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2461 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2521 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2581 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     2641 ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc taatttgtga
     2701 gtttagtata catgcattta cttataatac agttttttag ttttgctggc cgcatcttct
     2761 caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc
     2821 ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc
     2881 cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt
     2941 cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc
     3001 gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt
     3061 agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt
     3121 tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc
     3181 attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac
     3241 tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga
     3301 taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg
     3361 tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt
     3421 ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag
     3481 cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat
     3541 gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca
     3601 atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt
     3661 ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa
     3721 tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt
     3781 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     3841 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3901 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     3961 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagtatgat
     4021 ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga ggagtggcag
     4081 catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc atggaatggg
     4141 ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg catataagta
     4201 cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat atacaggcaa
     4261 cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt
     4321 tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc ctattctcta
     4381 gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag acgcactttc
     4441 aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa taccgcttcc
     4501 acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc cctatataac
     4561 ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta cattttttat
     4621 gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat tcatagagtg
     4681 aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga gacaaaatag
     4741 aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca ctttctgttc
     4801 acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt tatcttgaaa
     4861 aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc aggctttttt
     4921 tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga cctacagtgc
     4981 aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag taatctaaga
     5041 tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa aagaagtata
     5101 gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa aaatgcagct
     5161 cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta caaaaatgaa
     5221 gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc tgtaaaaatg
     5281 cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg ttctacaaaa
     5341 tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5401 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5461 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5521 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5581 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5641 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5701 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5761 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5821 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5881 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5941 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6001 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6061 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6121 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6181 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6241 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6301 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6361 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6421 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6481 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6541 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6601 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6661 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6721 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6781 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6841 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6901 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6961 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7021 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7081 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7141 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     7201 ggat
//
