LOCUS       sequence-402673-        6543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HCoV-229E 3CL protease from a GAL-3x promoter with a URA3 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6543)
  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 6543)
  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-3x-uC-HCoV-229E_3CL
FEATURES             Location/Qualifiers
     source          1..6543
                     /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"
     primer_bind     1447..1470
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1531..1555
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2531..2547)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2532..2548)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2550..2797
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2566..2584)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2815..2834)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2816..2834)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2844..2861)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2844..2860)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2853..2875)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3001..3456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3088..3107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3298..3319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3587..4390)
                     /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(4391..4606)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4630..4649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4749..4771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4809..4827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4868..5371
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5408..5512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5513..5581,5582..6373)
                     /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(5731..5750)
                     /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 tggagctccc gagcgggcga cagccctccg aaggaagact
     1081 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
     1141 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
     1201 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
     1261 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
     1321 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
     1381 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
     1441 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat tctagactga
     1501 accagatggc aaactagtgg atcccccatc acaagtttgt acaaaaaagc aggctacaaa
     1561 atggctggtt tgcgcaaaat ggcacaacca tctggctttg tggagaaatg tgttgtccgt
     1621 gtctgctatg gaaacactgt gttgaatggg ttgtggcttg gtgatattgt ttattgccca
     1681 cgtcatgtta tcgcatctaa cacaacttct gctatagatt atgatcacga atatagtatt
     1741 atgcggttgc ataatttttc tataatatct ggtacagcat ttcttggtgt tgtaggtgct
     1801 actatgcatg gagtaactct taaaattaag gtttcacaga ctaacatgca cacacctaga
     1861 cattctttta gaacactaaa atctggtgaa ggttttaaca tcttagcatg ctatgatggt
     1921 tgtgctcaag gtgtttttgg tgtgaacatg agaactaatt ggactatccg tggttcattt
     1981 attaatggtg cgtgtggttc ccctggctac aatcttaaaa atggcgaggt ggaatttgtt
     2041 tatatgcatc aaattgaact cggaagtggt agccatgtag gttctagctt tgatggtgtt
     2101 atgtatggtg gttttgaaga ccaacctaat cttcaagttg aatctgcaaa ccagatgtta
     2161 acagttaatg tggttgcatt tctttatgct gctatattga atggttgcac atggtggctt
     2221 aaaggtgaaa aattgtttgt ggagcattat aatgagtggg cacaggctaa tggtttcaca
     2281 gctatgaatg gtgaagacgc tttttccatt cttgctgcta aaactggtgt ctgtgtggaa
     2341 agattacttc atgctattca agttttgaat aatggctttg gtggtaaaca aattttgggt
     2401 tattctagtc tcaatgatga gttcagtatt aatgaagttg tcaaacaaat gtttggtgtt
     2461 aacctgcaat aaaacccagc tttcttgtac taagtggtga tgggctgcag gaattcgata
     2521 tcaagcttat cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc
     2581 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     2641 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     2701 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     2761 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat
     2821 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2881 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2941 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3001 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3061 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3421 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
     3481 gcatctgtgc ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc
     3541 taatttgtga gtttagtata catgcattta cttataatac agttttttag ttttgctggc
     3601 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag
     3661 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga
     3721 ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca
     3781 caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag
     3841 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat
     3901 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag
     3961 gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca
     4021 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact
     4081 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt
     4141 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga
     4201 tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta
     4261 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga
     4321 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag
     4381 ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga
     4441 atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc
     4501 tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaag
     4561 gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg tggtatggtg
     4621 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4681 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4741 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4801 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4861 ttagacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg atggaataat
     4921 ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt tagaaagtaa
     4981 ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat
     5041 ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga
     5101 tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc
     5161 tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa
     5221 aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat
     5281 tttttcttta atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat
     5341 ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg
     5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6541 ttc
//
