LOCUS       sequence-402864-        7189 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible destination vector for yeast expression with GAL1
            promoter (1x GAL4 binding sites)/6stop mutation/URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7189)
  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 7189)
  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-1x-ccdB-6stop
FEATURES             Location/Qualifiers
     source          1..7189
                     /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     1372..1395
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1416..1432
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1416..1432
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    1440..1564
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1589..1594,1595..1612,1613..1619)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1673..2332
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1739..1758)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2674..2979
                     /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     2858..2877
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(3177..3193)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3178..3194)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3196..3443
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3212..3230)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3461..3480)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3462..3480)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3490..3507)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3490..3506)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3499..3521)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3647..4102
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3734..3753)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3944..3965
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4233..5036)
                     /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(5037..5252)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5276..5295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5395..5417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5455..5473)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5514..6017
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6054..6158
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6159..6227,6228..7019)
                     /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(6377..6396)
                     /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 tggagctcgc agatgtgcct cgcgccgcac tgctccgaac
     1081 aataaagatt ctacaatact agcttttatg gttatgaaga ggaaaaattg gcagtaacct
     1141 ggccccacaa accttcaaat gaacgaatca aattaacaac cataggatga taatgcgatt
     1201 agttttttag ccttatttct ggggtaatta atcagcgaag cgatgatttt tgatctatta
     1261 acagatatat aaatgcaaaa actgcataac cactttaact aatactttca acattttcgg
     1321 tttgtattac ttcttattca aatgtaataa aagtatcaac aaaaaattgt taatatacct
     1381 ctatacttta acgtcaagga gaaaaaaccc cggattctag aactagtgga tcccccatca
     1441 caagtttgta caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     1501 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     1561 tatggcggcc gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt
     1621 gtggattttg agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa
     1681 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
     1741 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
     1801 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
     1861 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
     1921 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
     1981 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     2041 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     2101 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     2161 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     2221 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     2281 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aaacgcgtgg
     2341 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     2401 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     2461 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     2521 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     2581 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     2641 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     2701 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     2761 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     2821 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     2881 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     2941 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     3001 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     3061 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     3121 ttcagctttc ttgtactaag tggtgatggg ctgcaggaat tcgatatcaa gcttatcgat
     3181 accgtcgacc tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca
     3241 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
     3301 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
     3361 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
     3421 gacgctcgaa ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta
     3481 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3541 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3601 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc
     3661 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3721 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3781 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3841 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3901 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3961 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4021 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4081 aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4141 tttcacaccg catagggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     4201 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     4261 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     4321 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     4381 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     4441 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     4501 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     4561 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     4621 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     4681 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     4741 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     4801 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     4861 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     4921 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     4981 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     5041 tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     5101 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     5161 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa
     5221 aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt atggtgcact ctcagtacaa
     5281 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     5341 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5401 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5461 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acggatcgct
     5521 tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg gaatttactc
     5581 tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa agaaggtaga
     5641 agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca acaaaaagcg
     5701 tactttacat atatatttat tagacaagaa aagcagatta aatagatata cattcgatta
     5761 acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat
     5821 gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt agtatttgtt
     5881 ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt tctttaattt
     5941 ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa attataatta
     6001 tttttatagc acgtgatgaa aaggacccag gtggcacttt tcggggaaat gtgcgcggaa
     6061 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6121 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6181 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6241 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6301 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6361 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6421 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6481 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6541 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6601 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6661 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6721 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6781 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6841 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6901 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6961 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7021 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7081 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7141 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
