LOCUS       sequence-402865-        7576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible destination vector for yeast expression with 
            GAL10 promoter/6stop mutation/URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7576)
  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 7576)
  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-ccdB-6stop
FEATURES             Location/Qualifiers
     source          1..7576
                     /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..1951
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1976..1981,1982..1999,2000..2006)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2060..2719
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2126..2145)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3061..3366
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3245..3264
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(3564..3580)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3565..3581)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3583..3830
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3599..3617)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3848..3867)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3849..3867)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3877..3894)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3877..3893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3886..3908)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4034..4489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4331..4352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4620..5423)
                     /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(5424..5639)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5663..5682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5782..5804
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5842..5860)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5901..6404
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6441..6545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6546..6614,6615..7406)
                     /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(6764..6783)
                     /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 aaaagctgaa cgagaaacgt
     1861 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
     1921 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
     1981 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc
     2041 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     2101 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     2161 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     2221 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg
     2281 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     2341 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     2401 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     2461 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     2521 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     2581 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     2641 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     2701 gtggcagggc ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc
     2761 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
     2821 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
     2881 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
     2941 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
     3001 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
     3061 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     3121 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     3181 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     3241 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
     3301 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     3361 atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat
     3421 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     3481 gatatttata tcattttacg tttctcgttc agctttcttg tactaagtgg tgatgggctg
     3541 caggaattcg atatcaagct tatcgatacc gtcgacctcg agtcatgtaa ttagttatgt
     3601 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     3661 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     3721 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     3781 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc
     3841 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     3901 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3961 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4021 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     4501 attttctcct tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat
     4561 taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt
     4621 tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca
     4681 ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca
     4741 gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg
     4801 tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc
     4861 atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca
     4921 gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc
     4981 aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata
     5041 cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca
     5101 cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag
     5161 agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa
     5221 aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca
     5281 actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc
     5341 ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt
     5401 tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc
     5461 agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat
     5521 accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca
     5581 aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa
     5641 aggtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5701 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5761 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5821 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5881 taataatggt ttcttagacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta
     5941 atgatggaat aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga
     6001 ttttagaaag taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa
     6061 aggtttaaaa aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag
     6121 cagattaaat agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc
     6181 gtgtgtggtc ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga
     6241 agagcaagat aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga
     6301 aaacaaaaac tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt
     6361 tatattaaaa aatttaaatt ataattattt ttatagcacg tgatgaaaag gacccaggtg
     6421 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     6481 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6541 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6601 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6661 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6721 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6781 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6841 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6901 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6961 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     7021 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     7081 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     7141 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     7201 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     7261 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     7321 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     7381 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     7441 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     7501 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7561 agatcaaagg atcttc
//
