LOCUS       sequence-402863-        7261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible destination vector for yeast expression with GAL1
            promoter (2x GAL4 binding sites)/upstream ORF/6stop mutation/URA3 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7261)
  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 7261)
  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-ccdB-6stop
FEATURES             Location/Qualifiers
     source          1..7261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2754..2774
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     3172..3195
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    3256..3380
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3405..3410,3411..3428,3429..3435)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3489..4148
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3555..3574)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4490..4795
                     /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     4674..4693
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(4993..5009)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4994..5010)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5012..5259
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5028..5046)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5277..5296)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5278..5296)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5306..5323)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5306..5322)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5315..5337)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5463..5918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5550..5569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5760..5781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6049..6852)
                     /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(6853..7068)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7092..7111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7211..7233
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tcccgacgga agactctcct ccgtgcgtcc
     2821 tcgtcttcac cggtcgcgtt cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac
     2881 aataaagatt ctacaatact agcttttatg gttatgaaga ggaaaaattg gcagtaacct
     2941 ggccccacaa accttcaaat gaacgaatca aattaacaac cataggatga taatgcgatt
     3001 agttttttag ccttatttct ggggtaatta atcagcgaag cgatgatttt tgatctatta
     3061 acagatatat aaatgcaaaa actgcataac cactttaact aatactttca acattttcgg
     3121 tttgtattac ttcttattca aatgtaataa aagtatcaac aaaaaattgt taatatacct
     3181 ctatacttta acgtcaagga gaaaaaaccc cggattctag actgaacaag atggcaaact
     3241 agtggatccc ccatcacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata
     3301 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     3361 caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt
     3421 ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg
     3481 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     3541 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     3601 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     3661 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     3721 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
     3781 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     3841 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
     3901 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     3961 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     4021 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     4081 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
     4141 gggcgtaaac gcgtggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg
     4201 ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag
     4261 gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa
     4321 ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt
     4381 cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg
     4441 tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa
     4501 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     4561 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     4621 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     4681 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     4741 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     4801 aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt
     4861 tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat
     4921 cattttacgt ttctcgttca gctttcttgt actaagtggt gatgggctgc aggaattcga
     4981 tatcaagctt atcgataccg tcgacctcga gtcatgtaat tagttatgtc acgcttacat
     5041 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     5101 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     5161 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     5221 cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc aattcgccct
     5281 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5341 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5401 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5461 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5881 taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt
     5941 acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc
     6001 tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg
     6061 gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt
     6121 agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga
     6181 gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc
     6241 cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg
     6301 agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt
     6361 atattctcca gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct
     6421 aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac
     6481 cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta
     6541 ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt
     6601 gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa
     6661 gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag
     6721 taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat
     6781 gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt
     6841 agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa
     6901 gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag
     6961 tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca
     7021 aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg
     7081 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     7141 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     7201 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     7261 a
//
