LOCUS       sequence-402635-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HCoV-OC43 3CL 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 7378)
  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 7378)
  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-HCoV-OC43_3CL
FEATURES             Location/Qualifiers
     source          1..7378
                     /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(2480..2504)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2537..2553)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2538..2554)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2556..2803
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2572..2590)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2821..2840)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2822..2840)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2850..2867)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2850..2866)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2859..2881)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3007..3462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3094..3113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3304..3325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3593..4396)
                     /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(4397..4612)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4636..4655)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4755..4777
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4815..4833)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4874..6216
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5206..5253
                     /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        6243..6347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6348..6416,6417..7208)
                     /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(6566..6585)
                     /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 aaaatgtccg gcattgtgaa aatggtaaat cctacttcta aggtagaacc atgtgttgtc
     1621 agtgttacct atggtaatat gacattgaat ggtttatggt tggatgacaa ggtctactgt
     1681 cccagacatg taatatgttc tgcttcagat atgactaatc cagattatac aaatttgttg
     1741 tgtagagtaa catcaagtga ttttactgta ttgtttgatc gtctaagcct tacagtgatg
     1801 tcttatcaaa tgcggggttg tatgcttgtt cttacagtga ccctgcaaaa ttctcgtacg
     1861 ccaaaatata catttggtgt tgttaaacct ggtgagactt ttactgtttt agctgcttat
     1921 aacggcaaac cacaaggagc ctttcatgta actatgcgta gtagttatac cattaagggt
     1981 tcctttttat gcggatcttg tggatctgtt ggttatgtaa taatgggtga ttgtgttaaa
     2041 tttgtttata tgcatcaatt ggagcttagt actggttgtc atactggtac tgacttcaat
     2101 ggggattttt atggtcctta taaggatgct caggttgttc agttgcccat tcaggattat
     2161 atacaatctg ttaatttttt agcatggctt tatgctgcta tacttaacaa ttgtaattgg
     2221 tttatacaaa gtgataagtg ttctgtagaa gattttaatg tgtgggctct gtccaatgga
     2281 tttagccaag ttaaatctga ccttgttata gatgctttag cttctatgac tggtgtgtct
     2341 ttggaaacac tgttggctgc tattaagcgt cttaagaatg gtttccaagg acgtcagatt
     2401 atgggtagtt gctcttttga ggatgaattg acacctagcg atgtttatca acaactcgct
     2461 ggtatcaagt tacaataaaa cccagctttc ttgtacaaag tggtgatggg ctgcaggaat
     2521 tcgatatcaa gcttatcgat accgtcgacc tcgagtcatg taattagtta tgtcacgctt
     2581 acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga
     2641 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     2701 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     2761 cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt acccaattcg
     2821 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     2881 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2941 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3001 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3061 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     3541 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     3601 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     3661 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     3721 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     3781 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     3841 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     3901 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     3961 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     4021 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     4081 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     4141 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     4201 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     4261 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     4321 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     4381 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     4441 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     4501 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     4561 ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt
     4621 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4681 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4741 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4801 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4861 ggtttcttag tatgatccaa tatcaaagga aatgatagca ttgaaggatg agactaatcc
     4921 aattgaggag tggcagcata tagaacagct aaagggtagt gctgaaggaa gcatacgata
     4981 ccccgcatgg aatgggataa tatcacagga ggtactagac tacctttcat cctacataaa
     5041 tagacgcata taagtacgca tttaagcata aacacgcact atgccgttct tctcatgtat
     5101 atatatatac aggcaacacg cagatatagg tgcgacgtga acagtgagct gtatgtgcgc
     5161 agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac gctttgaagt tcctattccg
     5221 aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaaaac caaaagcgct
     5281 ctgaagacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct actaaaatat
     5341 tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat atctctgtgc
     5401 tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat ctaaactcga
     5461 cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa ttgtagtaag
     5521 aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct atacgtagta
     5581 tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg
     5641 ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga
     5701 tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt
     5761 ggagtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt
     5821 aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa
     5881 aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga
     5941 acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt
     6001 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     6061 gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt
     6121 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca
     6181 tttttgttct acaaaatgaa gcacagatgc ttcgttcagg tggcactttt cggggaaatg
     6241 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6301 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6361 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6421 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6481 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6541 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6601 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6661 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6721 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6781 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6841 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6901 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6961 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7021 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     7081 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7141 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7201 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7261 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7321 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
