LOCUS       sequence-402674-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Bat-CoV HKU4 3CL protease from a GAL10 promoter with a 
            URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  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 6851)
  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: pAG416GAL10-Bat-CoV-HKU4_3CL
FEATURES             Location/Qualifiers
     source          1..6851
                     /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        complement(1108..1549)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(1114..1137)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1432..1549
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    1827..1851
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2839..2855)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2840..2856)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2858..3105
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2874..2892)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3123..3142)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3124..3142)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3152..3169)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3152..3168)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3161..3183)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3309..3764
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3396..3415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3606..3627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3895..4698)
                     /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(4699..4914)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4938..4957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5057..5079
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5117..5135)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5176..5679
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5716..5820
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5821..5889,5890..6681)
                     /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(6039..6058)
                     /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 ggggtgtgtt
     1081 gttggtgggt tgggtgtata gttttttctc cttgacgtta aagtatagag gtatattaac
     1141 aattttttgt tgatactttt atgacatttg aataagaagt aatacaaact gaaaatgttg
     1201 aaagtattag ttaaagtggt tatgcagctt ttccatttat atatctgtta atagatcaaa
     1261 aatcatcgct tcgctgatta attaccccag aaataaggct aaaaaactaa tcgcattatc
     1321 atcctatggt tgttaatttg attcgttaat ttgaaggttt gtggggccag gttactgcca
     1381 atttttcctc ttcataacca taaaagctag tattgtagaa tctttattgt tcggagcagt
     1441 gcggcgcgag gcacatctgc gtttcaggaa cgcgaccggt gaagacgagg acgcacggag
     1501 gagagtcttc cgtcggaggg ctgtcgcccg ctcggcggct tctaatccgt acttcaatat
     1561 agcaatgagc agttaagcgt attactgaaa gttccaaaga gaaggttttt ttaggctaag
     1621 ataatggggc tctttacatt tccacaacat ataagtaaga ttagatatgg atatgtatat
     1681 ggtggtaatg ccatgtaata tgattattaa acttctttgc gtccatccaa aaaaaaagta
     1741 agaatttttg aaaattcaat ataaatgtct aaaggtgaag aattattcag aattccagct
     1801 gaccaccgac tagtggatcc cccatcacaa gtttgtacaa aaaagcaggc tacaaaatga
     1861 gtggtttggt taagatgtca gctcctagtg gtgctgtaga gaactgtatt gtgcaagtta
     1921 catgtgggtc tatgacattg aatggtcttt ggcttgataa cactgtatgg tgtcctcgcc
     1981 atattatgtg tcctgctgac cagttaactg acccaaatta tgatgctttg ttaatttcaa
     2041 aaactaatca tagttttatt gtgcaaaaac acataggagc tcaggctaat ctgcgtgttg
     2101 ttgctcattc tatggttggt gtcttactta agttaactgt tgatgttgct aatcctagta
     2161 caccagcata tacttttagt actgttaaac ctggcgcctc gtttagcgtt ttagcttgtt
     2221 ataatggcaa gcctacaggt gtctttacag ttaatttacg ccacaattct accattaaag
     2281 gcagtttcct ttgtggttct tgtggtagtg ttggttacac tgaaaatgga ggtgtaatca
     2341 actttgttta tatgcatcag atggaattat caaatggtac acatactggt agttcctttg
     2401 atggtgttat gtatggtgct tttgaagata aacaaacaca ccagttacag ttaacagaca
     2461 aatactgcac tattaatgta gtagcttggc tctatgcagc agttctaaat ggttgtaagt
     2521 ggtttgtaaa acccaccaga gttggaattg taacttataa tgaatgggct ttgagtaatc
     2581 agtttacaga atttgtaggc acacagtcca ttgatatgct tgctcaccga actggtgttt
     2641 ctgttgaaca aatgcttgct gctatccaga gtctgcatgc tggttttcag ggtaaaacta
     2701 ttctgggcca gtctactttg gaggatgagt tcactcctga tgatgttaat atgcaagtca
     2761 tgggtgttgt aatgcagtaa aacccagctt tcttgtacta agtggtgatg ggctgcagga
     2821 attcgatatc aagcttatcg ataccgtcga cctcgagtca tgtaattagt tatgtcacgc
     2881 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     2941 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     3001 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     3061 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt
     3121 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     3181 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3241 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3301 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3361 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3421 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3481 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3541 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3601 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3661 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3721 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     3781 ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat
     3841 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     3901 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     3961 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     4021 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     4081 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     4141 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     4201 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     4261 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     4321 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     4381 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     4441 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     4501 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     4561 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     4621 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     4681 atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg
     4741 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     4801 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     4861 atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg
     4921 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4981 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5041 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5101 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5161 atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat
     5221 ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta
     5281 gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt
     5341 taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat
     5401 taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg
     5461 tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc
     5521 aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca
     5581 aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat atatttatat
     5641 taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact
     5701 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5761 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5821 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5881 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5941 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6001 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6061 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6121 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6181 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6241 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6301 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6361 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6421 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6481 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6541 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6601 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6661 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6721 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6781 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6841 aaaggatctt c
//
