LOCUS       sequence-402648-        7351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HCoV-NL63 PLP2 from a GAL promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7351)
  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 7351)
  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: pAG426GAL-HCoV-NL63_PLP2
FEATURES             Location/Qualifiers
     source          1..7351
                     /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        1054..1495
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    1054..1171
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1466..1489
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1510..1526
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1510..1526
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1534..1558
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2453..2477)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2510..2526)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2511..2527)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2529..2776
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2545..2563)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2794..2813)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2795..2813)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2823..2840)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2823..2839)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2832..2854)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2980..3435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3067..3086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3277..3298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3566..4369)
                     /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(4370..4585)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4609..4628)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4728..4750
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4788..4806)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4847..6189
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5179..5226
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        6216..6320
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6321..6389,6390..7181)
                     /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(6539..6558)
                     /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 gtacggatta gaagccgccg agcgggtgac
     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 accccggatt ctagaactag tggatccccc atcacaagtt tgtacaaaaa agcaggctac
     1561 aaaatgctca agatcactga ggacggaatc aatgtaaagg atgtagtcgt ggaatcaagc
     1621 aagagtctgg ggaaacaact cggcgttgta agcgatgggg tggacagttt cgagggggtt
     1681 ctgcctatca acacggacac agttcttagc gtggccccgg aggtcgactg ggtagctttc
     1741 tacggcttcg aaaaagcggc cctgttcgcg tctttggacg taaaaccata tgggtatcca
     1801 aacgatttcg ttgggggctt tcgggttctc ggtaccaccg acaacaactg ttgggtcaac
     1861 gccacatgca tcattttgca atatctcaaa cctacattca aaagcaaggg tcttaatgta
     1921 ctttggaata aatttgtcac tggtgatgtt ggtccgttcg tttctttcat ttactttatc
     1981 accatgagca gtaaggggca gaagggcgat gccgaggaag cactgtctaa actctccgag
     2041 tacctgattt cagattccat cgtcacactg gaacaatata gtacttgcga catttgtaag
     2101 agcacagttg ttgaagttaa gtctgctatc gtctgcgcgt cagtgctgaa agatggctgt
     2161 gacgttggtt tctgtcctca ccgacacaag ctgaggtcaa gagtgaaatt tgtaaacggc
     2221 agagttgtaa ttactaatgt aggagaacca attatatctc agccgtctaa actcttgaac
     2281 gggattgctt acacgacctt ctcaggatcc tttgataatg gtcattatgt tgtctacgac
     2341 gctgcaaaca acgcagtgta tgatggagca agactgttca gttctgatct gtcaacattg
     2401 gcagtcacgg ctatcgtagt cgttggaggt tgtgtgacct caaatgtgta aaacccagct
     2461 ttcttgtaca aagtggtgat gggctgcagg aattcgatat caagcttatc gataccgtcg
     2521 acctcgagtc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc
     2581 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
     2641 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag
     2701 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
     2761 gaaggcttta atttgcggcc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     2821 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2881 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2941 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat
     3001 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3061 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3121 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3181 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3241 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3301 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3361 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3421 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3481 ccgcataggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac
     3541 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt
     3601 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata
     3661 gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat
     3721 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc
     3781 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat
     3841 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc
     3901 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg
     3961 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt
     4021 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa
     4081 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta
     4141 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta
     4201 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat
     4261 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa
     4321 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc
     4381 gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt
     4441 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt
     4501 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag
     4561 aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc actctcagta caatctgctc
     4621 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4681 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4741 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4801 cctattttta taggttaatg tcatgataat aatggtttct tagtatgatc caatatcaaa
     4861 ggaaatgata gcattgaagg atgagactaa tccaattgag gagtggcagc atatagaaca
     4921 gctaaagggt agtgctgaag gaagcatacg ataccccgca tggaatggga taatatcaca
     4981 ggaggtacta gactaccttt catcctacat aaatagacgc atataagtac gcatttaagc
     5041 ataaacacgc actatgccgt tcttctcatg tatatatata tacaggcaac acgcagatat
     5101 aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct
     5161 cgttttcgga aacgctttga agttcctatt ccgaagttcc tattctctag aaagtatagg
     5221 aacttcagag cgcttttgaa aaccaaaagc gctctgaaga cgcactttca aaaaaccaaa
     5281 aacgcaccgg actgtaacga gctactaaaa tattgcgaat accgcttcca caaacattgc
     5341 tcaaaagtat ctctttgcta tatatctctg tgctatatcc ctatataacc tacccatcca
     5401 cctttcgctc cttgaacttg catctaaact cgacctctac attttttatg tttatctcta
     5461 gtattactct ttagacaaaa aaattgtagt aagaactatt catagagtga atcgaaaaca
     5521 atacgaaaat gtaaacattt cctatacgta gtatatagag acaaaataga agaaaccgtt
     5581 cataattttc tgaccaatga agaatcatca acgctatcac tttctgttca caaagtatgc
     5641 gcaatccaca tcggtataga atataatcgg ggatgccttt atcttgaaaa aatgcacccg
     5701 cagcttcgct agtaatcagt aaacgcggga agtggagtca ggcttttttt atggaagaga
     5761 aaatagacac caaagtagcc ttcttctaac cttaacggac ctacagtgca aaaagttatc
     5821 aagagactgc attatagagc gcacaaagga gaaaaaaagt aatctaagat gctttgttag
     5881 aaaaatagcg ctctcgggat gcatttttgt agaacaaaaa agaagtatag attctttgtt
     5941 ggtaaaatag cgctctcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg
     6001 tttgaaaaat tagcgctctc gcgttgcatt tttgttttac aaaaatgaag cacagattct
     6061 tcgttggtaa aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc agctcagatt
     6121 ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tctacaaaat gaagcacaga
     6181 tgcttcgttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6241 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6301 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6361 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6421 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6481 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6541 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6601 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6661 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6721 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6781 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6841 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6901 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6961 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     7021 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7081 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7141 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7201 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7261 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7321 cagaccccgt agaaaagatc aaaggatctt c
//
