LOCUS       sequence-402633-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SLEV NS2B-NS3 protease (with GS linker) from a GAL 
            promoter with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  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 7204)
  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-SLEV_NS2B-GS-NS3
FEATURES             Location/Qualifiers
     source          1..7204
                     /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(2306..2330)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2363..2379)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2364..2380)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      2382..2629
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2398..2416)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2647..2666)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2648..2666)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2676..2693)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2676..2692)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2685..2707)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2833..3288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2920..2939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3130..3151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3419..4222)
                     /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(4223..4438)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4462..4481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4581..4603
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4641..4659)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      4700..6042
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    5032..5079
                     /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        6069..6173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6174..6242,6243..7034)
                     /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(6392..6411)
                     /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 aaaatggtct caggaaaagc cgcagaaatg tggattgaaa aagcagcaga catcacatgg
     1621 gaacagaatg ccgagatcac cggaaccagc cccaggcttg atgttgacct tgactcacat
     1681 ggcaacttca agctcctgaa cgaccctgga gctccagtgc acggaggtgg aggttcaggg
     1741 gggggtggag gaggagcatt gtgggacgtg ccgtcaccaa aagtgtatcc aaagtgtgag
     1801 acaaaaccag gaatctatcg aatcatgact agaggaattt tgggcacttt ccaagccgga
     1861 gtcggtgtga tgcacgaagg agtttttcac acaatgtggc acgcaactga aggagcagtc
     1921 ctacgaaatg gagaaggaag actagatccg tatgcaggag atgtccggaa tgatctgatc
     1981 tcgtatggag gtccctggaa gctgagtgcc acttgggatg gaactgagga ggtccaaatg
     2041 attgcagtag ctccggggaa accagccatt aatgtccaga caacaccagg agtgtttaag
     2101 accccgctcg ggaccattgg agccgtgacg cttgatttcc caaaaggaac atcaggttct
     2161 ccaattatca acaagaaagg ggaaatcatt gggctctatg gaaatggggt attgattgga
     2221 cagggcgaat atgttagtgg catcatccaa ggggaaagga cagaagaacc aattccagat
     2281 gcatacaatg aggaaatgct ctaaaaccca gctttcttgt acaaagtggt gatgggctgc
     2341 aggaattcga tatcaagctt atcgataccg tcgacctcga gtcatgtaat tagttatgtc
     2401 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca
     2461 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt
     2521 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact
     2581 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc
     2641 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     2701 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2761 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2821 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2881 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2941 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3001 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3061 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3121 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3181 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3241 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
     3301 ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt
     3361 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt
     3421 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac
     3481 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag
     3541 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt
     3601 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca
     3661 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag
     3721 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca
     3781 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac
     3841 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac
     3901 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga
     3961 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa
     4021 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa
     4081 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct
     4141 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt
     4201 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca
     4261 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata
     4321 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa
     4381 aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa
     4441 ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4501 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4561 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4621 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4681 aataatggtt tcttagtatg atccaatatc aaaggaaatg atagcattga aggatgagac
     4741 taatccaatt gaggagtggc agcatataga acagctaaag ggtagtgctg aaggaagcat
     4801 acgatacccc gcatggaatg ggataatatc acaggaggta ctagactacc tttcatccta
     4861 cataaataga cgcatataag tacgcattta agcataaaca cgcactatgc cgttcttctc
     4921 atgtatatat atatacaggc aacacgcaga tataggtgcg acgtgaacag tgagctgtat
     4981 gtgcgcagct cgcgttgcat tttcggaagc gctcgttttc ggaaacgctt tgaagttcct
     5041 attccgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt gaaaaccaaa
     5101 agcgctctga agacgcactt tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta
     5161 aaatattgcg aataccgctt ccacaaacat tgctcaaaag tatctctttg ctatatatct
     5221 ctgtgctata tccctatata acctacccat ccacctttcg ctccttgaac ttgcatctaa
     5281 actcgacctc tacatttttt atgtttatct ctagtattac tctttagaca aaaaaattgt
     5341 agtaagaact attcatagag tgaatcgaaa acaatacgaa aatgtaaaca tttcctatac
     5401 gtagtatata gagacaaaat agaagaaacc gttcataatt ttctgaccaa tgaagaatca
     5461 tcaacgctat cactttctgt tcacaaagta tgcgcaatcc acatcggtat agaatataat
     5521 cggggatgcc tttatcttga aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcg
     5581 ggaagtggag tcaggctttt tttatggaag agaaaataga caccaaagta gccttcttct
     5641 aaccttaacg gacctacagt gcaaaaagtt atcaagagac tgcattatag agcgcacaaa
     5701 ggagaaaaaa agtaatctaa gatgctttgt tagaaaaata gcgctctcgg gatgcatttt
     5761 tgtagaacaa aaaagaagta tagattcttt gttggtaaaa tagcgctctc gcgttgcatt
     5821 tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc
     5881 atttttgttt tacaaaaatg aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt
     5941 gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
     6001 gttgcatttt tgttctacaa aatgaagcac agatgcttcg ttcaggtggc acttttcggg
     6061 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6121 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6181 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6241 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6301 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6361 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6421 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6481 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6541 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6601 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6661 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6721 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6781 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6841 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6901 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6961 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7021 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7081 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7141 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7201 cttc
//
