LOCUS       sequence-402653-        6546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HCoV-NL63 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 6546)
  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 6546)
  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-NL63_3CL
FEATURES             Location/Qualifiers
     source          1..6546
                     /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(2534..2550)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2535..2551)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2553..2800
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2569..2587)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2818..2837)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2819..2837)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2847..2864)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2847..2863)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2856..2878)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3004..3459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3091..3110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3301..3322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3590..4393)
                     /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(4394..4609)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4633..4652)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4752..4774
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4812..4830)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4871..5374
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5411..5515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5516..5584,5585..6376)
                     /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(5734..5753)
                     /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 atgtctggtc ttaagaagat ggcacaacca tctggttgtg ttgagagatg tgtggttcgc
     1621 gtctgttatg gtagtactgt gcttaatgga gtttggttag gtgacactgt tacttgtcct
     1681 agacatgtca tagcaccatc aaccactgtt cttattgatt atgatcatgc atatagtact
     1741 atgcgtttgc ataatttttc agtgtcttat aatggtgtct tcttgggagt tgttggtgtt
     1801 acaatgcatg gttctgtgtt gcgtattaag gtttcacaat ctaatgtaca tacacctaaa
     1861 catgttttta aaacgttgaa acctggtgat tcttttaata ttttagcatg ttatgaaggt
     1921 attgcatctg gtgtttttgg tgttaattta cgtacaaact ttactattaa aggttctttt
     1981 ataaatggag cttgtggttc tcctggttat aatgttagaa atgatggtac tgttgagttt
     2041 tgttatttac accaaattga gttaggtagt ggtgctcatg ttggttctga ttttactggt
     2101 agtgtttatg gtaattttga tgaccaacct agtttgcaag ttgagagtgc caaccttatg
     2161 ctatcagata atgttgttgc ctttttgtat gctgctttgt tgaatggttg taggtggtgg
     2221 ttgcgttcaa ctagagttaa tgttgatggt tttaatgaat gggctatggc taatggttat
     2281 acaagtgttt ctagtgttga gtgctattct attttggcag caaaaactgg tgttagtgtt
     2341 gaacaattgt tagcttccat tcaacatctt catgaaggtt ttggtggtaa aaacatactt
     2401 ggttattcta gtttatgtga tgagttcaca ctagctgaag ttgtgaagca gatgtatggt
     2461 gttaacttgc aataaaaccc agctttcttg tactaagtgg tgatgggctg caggaattcg
     2521 atatcaagct tatcgatacc gtcgacctcg agtcatgtaa ttagttatgt cacgcttaca
     2581 ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
     2641 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
     2701 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
     2761 gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc
     2821 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     2881 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2941 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     3001 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3061 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     3481 tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag
     3541 ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct
     3601 ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct
     3661 tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag
     3721 agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac
     3781 ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt
     3841 gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag
     3901 tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc
     3961 taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca
     4021 ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt
     4081 actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat
     4141 tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca
     4201 agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca
     4261 gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca
     4321 tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg
     4381 tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta
     4441 agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa
     4501 gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc
     4561 aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg
     4621 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4681 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4741 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4801 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4861 ttcttagacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
     4921 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
     4981 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
     5041 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
     5101 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
     5161 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
     5221 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
     5281 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
     5341 aatttaaatt ataattattt ttatagcacg tgatgaaaag gacccaggtg gcacttttcg
     5401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5881 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5941 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6001 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6061 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6121 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6181 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6241 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6301 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6361 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6421 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6481 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6541 atcttc
//
