LOCUS       sequence-402859-        6609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses DENV 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 6609)
  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 6609)
  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: pAG416GAL-DENV_NS3-GS-NS2B
FEATURES             Location/Qualifiers
     source          1..6609
                     /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(2540..2564)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2597..2613)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2598..2614)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2616..2863
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2632..2650)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2881..2900)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2882..2900)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2910..2927)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2910..2926)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2919..2941)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3067..3522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3154..3173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3364..3385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3653..4456)
                     /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(4457..4672)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4696..4715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4815..4837
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4875..4893)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4934..5437
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5474..5578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5579..5647,5648..6439)
                     /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(5797..5816)
                     /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 aaaatgtcag gagtgctatg ggacacaccc agccctccag aagtggaaag agcagtcctt
     1621 gatgatggca tttatagaat tctccaaaga ggattgttgg gcaggtctca agtaggagta
     1681 ggagtttttc aagaaggcgt gttccacaca atgtggcacg tcaccagggg agctgtcctc
     1741 atgtaccaag ggaagagact ggaaccaagt tgggccagtg tcaaaaaaga cttgatctca
     1801 tatggaggag gttggaggtt tcaaggatcc tggaacgcgg gagaagaagt gcaggtgatt
     1861 gctgttgaac cggggaagaa ccccaaaaat gtacagacag cgccgggtac cttcaagacc
     1921 cctgaaggcg aagttggagc catagctcta gactttaaac ccggcacatc tggatctcct
     1981 atcgtgaaca gagagggaaa aatagtaggt ctttatggaa atggagtggt gacaacaagt
     2041 ggtacctacg tcagtgccat agctcaagct aaagcatcac aagaagggcc tctaccagag
     2101 attgaggacg aggtgtttgg aggtggaggt tcaggaggtg gtggaagctg gcctctcaat
     2161 gaaggaatta tggctgttgg aatagttagc attcttctaa gttcacttct caagaatgat
     2221 gtgccactag ctggcccact aatagctgga ggcatgctaa tagcatgtta tgtcatatct
     2281 ggaagctcgg ccgatttatc actggagaaa gcggctgagg tctcctggga agaagaagca
     2341 gaacactctg gtgcctcaca caacatacta gtggaggtcc aagatgatgg aaccatgaag
     2401 ataaaggatg aagagagaga tgacacactc accattctcc tcaaagcaac tctgctagca
     2461 atctcagggg tatacccaat gtcaataccg gcgaccctct ttgtgtggta tttttggcag
     2521 aaaaagaaac agagataaaa cccagctttc ttgtacaaag tggtgatggg ctgcaggaat
     2581 tcgatatcaa gcttatcgat accgtcgacc tcgagtcatg taattagtta tgtcacgctt
     2641 acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga
     2701 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     2761 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     2821 cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt acccaattcg
     2881 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     2941 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3001 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3061 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3121 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3181 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3241 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3301 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3361 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3421 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3481 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     3541 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     3601 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     3661 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     3721 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     3781 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     3841 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     3901 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     3961 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     4021 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     4081 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     4141 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     4201 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     4261 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     4321 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     4381 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     4441 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     4501 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     4561 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     4621 ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt
     4681 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4741 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4801 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4861 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4921 ggtttcttag acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg
     4981 aataatttgg gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga
     5041 aagtaaataa agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta
     5101 aaaaatttca acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta
     5161 aatagatata cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg
     5221 gtcttctaca cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa
     5281 gataaaaggt agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa
     5341 aactattttt tctttaattt ctttttttac tttctatttt taatttatat atttatatta
     5401 aaaaatttaa attataatta tttttatagc acgtgatgaa aaggacccag gtggcacttt
     5461 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5521 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5761 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5881 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6121 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6181 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6421 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6481 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6541 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6601 aggatcttc
//
