LOCUS       sequence-402751-        6527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Beluga Whale-CoV SW1 3CL protease from a GAL-2x promoter 
            with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6527)
  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 6527)
  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-2x-uA-BWCoV-SW1_3CL
FEATURES             Location/Qualifiers
     source          1..6527
                     /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     1428..1451
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1512..1536
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2515..2531)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2516..2532)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2534..2781
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2550..2568)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2799..2818)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2800..2818)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2828..2845)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2828..2844)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2837..2859)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2985..3440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3072..3091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3282..3303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3571..4374)
                     /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(4375..4590)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4614..4633)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4733..4755
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4793..4811)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4852..5355
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5392..5496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5497..5565,5566..6357)
                     /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(5715..5734)
                     /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 gacggaagac tctcctccgt gcgtcctcgt
     1081 cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata
     1141 aagattctac aatactagct tttatggtta tgaagaggaa aaattggcag taacctggcc
     1201 ccacaaacct tcaaatgaac gaatcaaatt aacaaccata ggatgataat gcgattagtt
     1261 ttttagcctt atttctgggg taattaatca gcgaagcgat gatttttgat ctattaacag
     1321 atatataaat gcaaaaactg cataaccact ttaactaata ctttcaacat tttcggtttg
     1381 tattacttct tattcaaatg taataaaagt atcaacaaaa aattgttaat atacctctat
     1441 actttaacgt caaggagaaa aaaccccgga ttctagactg aacaagatgg caaactagtg
     1501 gatcccccat cacaagtttg tacaaaaaag caggctacaa aatggcaggt ataaagaaga
     1561 tggtcgctcc ttcatctgct gtagaacagt gtgtagtatc tgttgtgcat ggtaacacac
     1621 agcttaatgg tttgtggttg aatgattatg tgttatgccc tcgtcatata cttggtaagt
     1681 atactggtga gcaatggagg gatgcactta ttaatgctaa taattttgat ttccatattt
     1741 tgtataaggg tatggaactt caagttgtgg gccgtgaatt ggtaggcgca cttttaaaat
     1801 tgaaagtttc tatggtaaat gctaatacac ctaagtataa gtttgccaaa gctagaattg
     1861 gtgataactt ttccattgct tgtgcttata atggacatgt ttctggtttg tacactgtca
     1921 cactccgaga aaatggaaca cttaaaggta gtttcatgtc aggatcgtgt ggtagtgttg
     1981 gttataatgt gactaatgaa ggtgttgaat ttgtttatat gcaccatttg gaacttccag
     2041 ggtgcgtgca tggtggtagt gaccttcatg gcatatttta tggcggttat gtagacgaag
     2101 aagtacttca gaggatacct cctgcaccag ctaattcacg taatattgtt gcgtggttgt
     2161 atgctgcagt ctataataac tgtgattggt ttgtgaaata tggacctaag caagttatgt
     2221 ctgtagaaga ttttaatgag tgggcatctg gatatggttt taccaaattt gagtaccacc
     2281 ttgcttttga tgtgttttca gcagcaactg gagtttctgt ggagcagatg cttgcagcca
     2341 taaaggagtt ggccgatggg tggaattatg cccctgtttt gggttcattc catcttgatg
     2401 atgaatattc tcctgaaatg attatgcaac aaactagtgg cattgttcta cagtaaaacc
     2461 cagctttctt gtactaagtg gtgatgggct gcaggaattc gatatcaagc ttatcgatac
     2521 cgtcgacctc gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     2581 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     2641 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     2701 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     2761 cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg
     2821 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     2881 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     2941 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     3001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3061 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3181 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3301 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3361 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3421 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3481 tcacaccgca tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag
     3541 tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata
     3601 tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc
     3661 aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt
     3721 tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat
     3781 caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac
     3841 aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag
     3901 ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc
     3961 ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt
     4021 aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt
     4081 accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc
     4141 ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt
     4201 tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg
     4261 aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc
     4321 agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta
     4381 tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca
     4441 tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg
     4501 ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa
     4561 aaaagaataa aaaaaaaatg atgaattgaa aaggtggtat ggtgcactct cagtacaatc
     4621 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4681 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4741 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4801 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac ggatcgcttg
     4861 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
     4921 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
     4981 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
     5041 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
     5101 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
     5161 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
     5221 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
     5281 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
     5341 tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc
     5401 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5461 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5521 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5581 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5641 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5701 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5761 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5821 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5881 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5941 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6001 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6061 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6121 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6181 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6241 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6301 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6361 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6421 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6481 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
