LOCUS       sequence-402866-        7280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible destination vector for yeast expression with GAL1
            promoter (3x 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 7280)
  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 7280)
  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-3x-uC-ccdB-6stop
FEATURES             Location/Qualifiers
     source          1..7280
                     /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     1447..1470
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    1531..1655
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1680..1685,1686..1703,1704..1710)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1764..2423
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1830..1849)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2765..3070
                     /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     2949..2968
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(3268..3284)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3269..3285)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3287..3534
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3303..3321)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3552..3571)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3553..3571)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3581..3598)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3581..3597)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3590..3612)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3738..4193
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3825..3844)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4035..4056
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4324..5127)
                     /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(5128..5343)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5367..5386)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5486..5508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5546..5564)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5605..6108
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6145..6249
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6250..6318,6319..7110)
                     /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(6468..6487)
                     /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 tggagctccc gagcgggcga cagccctccg aaggaagact
     1081 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
     1141 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
     1201 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
     1261 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
     1321 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
     1381 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
     1441 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat tctagactga
     1501 accagatggc aaactagtgg atcccccatc acaagtttgt acaaaaaagc tgaacgagaa
     1561 acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa acagactaca
     1621 taatactgta aaacacaaca tatccagtca ctatggcggc cgcattaggc accccaggct
     1681 ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat ccgtcgagat
     1741 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     1801 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     1861 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     1921 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     1981 tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     2041 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     2101 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     2161 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     2221 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     2281 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     2341 atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     2401 atgagtggca gggcggggcg taaacgcgtg gatccggctt actaaaagcc agataacagt
     2461 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     2521 gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     2581 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     2641 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     2701 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg
     2761 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     2821 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     2881 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     2941 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     3001 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     3061 gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact
     3121 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     3181 tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtactaa gtggtgatgg
     3241 gctgcaggaa ttcgatatca agcttatcga taccgtcgac ctcgagtcat gtaattagtt
     3301 atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt
     3361 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     3421 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     3481 atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg
     3541 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3601 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3661 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3721 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3781 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3841 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3901 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3961 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4021 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4081 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4141 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     4201 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagggta ataactgata
     4261 taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt
     4321 tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg
     4381 ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat
     4441 gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc
     4501 cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc
     4561 acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc
     4621 aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa
     4681 catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac
     4741 aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta
     4801 tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc
     4861 gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat
     4921 ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc
     4981 ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt
     5041 ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc
     5101 acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct
     5161 gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat
     5221 atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga
     5281 atcaaaaaaa tttcaaggaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt
     5341 gaaaaggtgg tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5401 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5461 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5521 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5581 atgataataa tggtttctta gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct
     5641 tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt
     5701 tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa
     5761 acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga
     5821 aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa atcacaggat
     5881 tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat acctgagagc
     5941 aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct tttacatctt
     6001 cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt ttaatttata
     6061 tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga aaaggaccca
     6121 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     6181 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     6241 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6301 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6361 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6421 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6481 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6541 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6601 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6661 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6721 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6781 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6841 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6901 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6961 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     7021 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     7081 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     7141 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     7201 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     7261 gaaaagatca aaggatcttc
//
