LOCUS       sequence-402652-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CV-B3 3C protease from a GAL promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  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 7008)
  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-CV-B3_3C
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     350..367
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    484..505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(520..525,526..543,544..550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    558..574
                     /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     563..585
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     582..598
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     582..598
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     617..637
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        619..637
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        661..1102
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    661..778
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1073..1096
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1117..1133
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1117..1133
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1141..1165
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1727..1751)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1784..1800)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1785..1801)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      1803..2050
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1819..1837)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2068..2087)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2069..2087)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2097..2114)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2097..2113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2106..2128)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2254..2709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2341..2360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2551..2572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2840..3643)
                     /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(3644..3859)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3883..3902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4002..4024
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4062..4080)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4121..5463
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    4453..4500
                     /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        5490..5594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5595..5663,5664..6455)
                     /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(5813..5832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
       61 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      121 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      181 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      241 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      301 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      361 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      421 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      481 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      541 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      601 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctctagta
      661 cggattagaa gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc
      721 ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa
      781 caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc
      841 tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat
      901 tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt
      961 aacagatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg
     1021 gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc
     1081 tctatacttt aacgtcaagg agaaaaaacc ccggattcta gaactagtgg atcccccatc
     1141 acaagtttgt acaaaaaagc aggctacaaa atgggccctg cctttgagtt cgctgtcgca
     1201 atgatgaaaa ggaactcaag cagggtgaaa actgaatatg gcgagtttac catgctgggc
     1261 atctatgaca ggtgggccgt tttgccacgc cacgccaaac ctgggccaac catcttgatg
     1321 aatgatcaag aggttggtgt gctagatgcc aaggagctag tagacaagga cggcaccaac
     1381 ttagaactga cactactcaa attgaaccgg aatgagaagt tcagagacat cagaggcttc
     1441 ttagccaagg aggaagtgga ggttaatgag gcagtgctag caattaacac cagcaagttt
     1501 cccaacatgt acattccagt aggacaggtc acagaatacg gcttcttaaa cctaggtggc
     1561 acacccacca agagaatgct tatgtacaac ttccccacaa gagcaggcca gtgtggtgga
     1621 gtgctcatgt ccaccggcaa ggtactgggt atccatgttg gtggaaatgg ccatcagggc
     1681 ttctcagcag cactcctcaa acactacttc aatgatgagc aataaaaccc agctttcttg
     1741 tacaaagtgg tgatgggctg caggaattcg atatcaagct tatcgatacc gtcgacctcg
     1801 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     1861 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     1921 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     1981 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     2041 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2101 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2161 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2221 tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg
     2281 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2341 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2401 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2461 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2521 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2581 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2641 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2701 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2761 agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat
     2821 ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc
     2881 ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc
     2941 ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt
     3001 gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt
     3061 aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt
     3121 cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc
     3181 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct
     3241 gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc
     3301 attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag
     3361 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct
     3421 taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa
     3481 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg
     3541 aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac
     3601 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc
     3661 tgcaggtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa
     3721 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct
     3781 gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa
     3841 aaaaaaatga tgaattgaaa aggtggtatg gtgcactctc agtacaatct gctctgatgc
     3901 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3961 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4021 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4081 tttataggtt aatgtcatga taataatggt ttcttagtat gatccaatat caaaggaaat
     4141 gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa
     4201 gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt
     4261 actagactac ctttcatcct acataaatag acgcatataa gtacgcattt aagcataaac
     4321 acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag atataggtgc
     4381 gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt
     4441 cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
     4501 agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac caaaaacgca
     4561 ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca ttgctcaaaa
     4621 gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca tccacctttc
     4681 gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc tctagtatta
     4741 ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa aacaatacga
     4801 aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac cgttcataat
     4861 tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt atgcgcaatc
     4921 cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca cccgcagctt
     4981 cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa gagaaaatag
     5041 acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt tatcaagaga
     5101 ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg ttagaaaaat
     5161 agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt tgttggtaaa
     5221 atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa
     5281 aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg
     5341 gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt
     5401 ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc
     5461 gttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5821 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6121 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctac
//
