LOCUS       sequence-402845-        6574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 3CL protease from a GALL promoter with a URA3 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6574)
  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 6574)
  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: pAG416GALL-SARS-CoV-2_3CL
FEATURES             Location/Qualifiers
     source          1..6574
                     /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     1466..1489
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1550..1574
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2562..2578)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2563..2579)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2581..2828
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2597..2615)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2846..2865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2847..2865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2875..2892)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2875..2891)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2884..2906)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3032..3487
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3119..3138)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3329..3350
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3618..4421)
                     /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(4422..4637)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4661..4680)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4780..4802
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4840..4858)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4899..5402
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5439..5543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5544..5612,5613..6404)
                     /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(5762..5781)
                     /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 tgaccatgat tacgccaagc ggctcgtgac
     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 accccggatc cggaattcca gctgaccacc gactagtgga tcccccatca caagtttgta
     1561 caaaaaagca ggctacaaaa tgagtggttt tagaaaaatg gcattcccat ctggtaaagt
     1621 tgagggttgt atggtacaag taacttgtgg tacaactaca cttaacggtc tttggcttga
     1681 tgacgtagtt tactgtccaa gacatgtgat ctgcacctct gaagacatgc ttaaccctaa
     1741 ttatgaagat ttactcattc gtaagtctaa tcataatttc ttggtacagg ctggtaatgt
     1801 tcaactcagg gttattggac attctatgca aaattgtgta cttaagctta aggttgatac
     1861 agccaatcct aagacaccta agtataagtt tgttcgcatt caaccaggac agactttttc
     1921 agtgttagct tgttacaatg gttcaccatc tggtgtttac caatgtgcta tgaggcccaa
     1981 tttcactatt aagggttcat tccttaatgg ttcatgtggt agtgttggtt ttaacataga
     2041 ttatgactgt gtctcttttt gttacatgca ccatatggaa ttaccaactg gagttcatgc
     2101 tggcacagac ttagaaggta acttttatgg accttttgtt gacaggcaaa cagcacaagc
     2161 agctggtacg gacacaacta ttacagttaa tgttttagct tggttgtacg ctgctgttat
     2221 aaatggagac aggtggtttc tcaatcgatt taccacaact cttaatgact ttaaccttgt
     2281 ggctatgaag tacaattatg aacctctaac acaagaccat gttgacatac taggacctct
     2341 ttctgctcaa actggaattg ccgttttaga tatgtgtgct tcattaaaag aattactgca
     2401 aaatggtatg aatggacgta ccatattggg tagtgcttta ttagaagatg aatttacacc
     2461 ttttgatgtt gttagacaat gctcaggtgt tactttccaa taaaacccag ctttcttgta
     2521 ctaagtggtg atgggctgca ggaattcgat atcaagctta tcgataccgt cgacctcgag
     2581 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg
     2641 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     2701 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     2761 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt
     2821 taatttgcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     2881 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2941 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3001 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
     3061 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3121 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3181 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3241 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3301 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3361 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3421 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3481 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag
     3541 ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt
     3601 acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct
     3661 gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt
     3721 ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga
     3781 cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa
     3841 ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg
     3901 ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat
     3961 gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc
     4021 ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat
     4081 tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca
     4141 aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta
     4201 actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt
     4261 ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa
     4321 gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta
     4381 ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg
     4441 caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca
     4501 ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt
     4561 tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa
     4621 aaaaatgatg aattgaaaag gtggtatggt gcactctcag tacaatctgc tctgatgccg
     4681 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4741 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4801 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4861 tataggttaa tgtcatgata ataatggttt cttagacgga tcgcttgcct gtaacttaca
     4921 cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt
     4981 ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga
     5041 agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata
     5101 tttattagac aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg
     5161 taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat
     5221 taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga
     5281 gtcttttaca tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct
     5341 atttttaatt tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg
     5401 atgaaaagga cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5461 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5521 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5581 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5641 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5701 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5761 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5821 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5881 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5941 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6001 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6061 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6121 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6181 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6241 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6301 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6361 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6421 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6481 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6541 cgtcagaccc cgtagaaaag atcaaaggat cttc
//
