LOCUS       sequence-420486-        7550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-inducible expression of NUbo-E3(48)-VSV in budding 
            yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7550)
  AUTHORS   Renz C, Asimaki E, Meister C, Albanese V, Petriukov K, Krapoth NC, 
            Wegmann S, Wollscheid HP, Wong RP, Fulzele A, Chen JX, Leon S, 
            Ulrich HD
  TITLE     Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
  JOURNAL   Mol Cell. 2024 Jan 18;84(2):386-400.e11. doi: 
            10.1016/j.molcel.2023.11.016. Epub 2023 Dec 15.
  PUBMED    38103558
REFERENCE   2  (bases 1 to 7550)
  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: 
            "10.1016/j.molcel.2023.11.016"; journalName: "Mol Cell"; date: 
            "2024-01-18- 18"; volume: "84"; issue: "2"; pages: "386-400.e11"
COMMENT     Sequence Label: YIp211-TET-NUbo-E3(48)-VSV
FEATURES             Location/Qualifiers
     source          1..7550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..69)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(223..811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(303..322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(982..1773,1774..1842))
                     /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     1605..1624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1843..1947)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2015..2033
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2040..2255
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2256..3059
                     /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"
     primer_bind     complement(3177..3199)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3299..3318
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(3392..3625)
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             complement(3893..4513)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLKGESWQDFLRNNAKSFRCALLSHRNGAKVHSDTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4421..4440
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(4593..4617)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4614..4817)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4643..4663)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4818..5197
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     terminator      5309..5496
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5364..5388)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    join(5554..5572,5573..5595,5596..5614,5615..5637,5638..5656,
                     5657..5679,5680..5698,5699..5721,5722..5740,5741..5763,
                     5764..5782,5783..5805,5806..5824)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             6140..6166
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6176..6454
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             7016..7048
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      7083..7330
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7099..7117)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7371..7387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7371..7387)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7384..7406)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7395..7411
                     /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)."
     promoter        complement(join(7419..7425,7426..7443,7444..7449))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7464..7485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
       61 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
      121 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      181 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      241 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      301 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      361 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      421 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      481 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      541 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      601 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      661 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      721 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      781 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      841 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      901 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      961 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1021 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     1081 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     1141 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1201 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1261 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1321 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1381 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1441 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1501 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1561 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     1621 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1681 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1741 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1801 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1861 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1921 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     1981 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccagcttt
     2041 tcaattcatc attttttttt tattcttttt tttgatttcg gtttccttga aatttttttg
     2101 attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt agattggtat
     2161 atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta acccaactgc
     2221 acagaacaaa aacatgcagg aaacgaagat aaatcatgtc gaaagctaca tataaggaac
     2281 gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg cacgaaaagc
     2341 aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg gagttagttg
     2401 aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg actgattttt
     2461 ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat tttttactct
     2521 tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac tctgcgggtg
     2581 tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg ggcccaggta
     2641 ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga ggccttttga
     2701 tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact aagggtactg
     2761 ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa agagacatgg
     2821 gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt ttagatgaca
     2881 agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct acaggatctg
     2941 acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag gtagagggtg
     3001 aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag caaaactaaa
     3061 aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat
     3121 tatatcagtt attacccgcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     3181 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     3241 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     3301 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     3361 gtaaggagaa aataccgcat caggcgccct acccaccgta ctcgtcaatt ccaagggcat
     3421 cggtaaacat ctgctcaaac tcgaagtcgg ccatatccag agcgccgtag ggggcggagt
     3481 cgtggggggt aaatcccgga cccggggaat ccccgtcccc caacatgtcc agatcgaaat
     3541 cgtctagcgc gtcggcatgc gccatcgcca cgtcctcgcc gtctaagtgg agctcgtccc
     3601 ccaggctgac atcggtcggg ggggccgtcg acagtctgcg cgtgtgtccc gcggggagaa
     3661 aggacaggcg cggagccgcc agccccgcct cttcgggggc gtcgtcgtcc gggagatcga
     3721 gcaggccctc gatggtagac ccgtaattgt ttttcgtacg cgcggtaccg cttacccatc
     3781 tctccgcatc acctttggta aaggttctaa gcttaggtga gaacatccct gcctgaacat
     3841 gagaaaaaac agggtactca tactcacttc taagtgacgg ctgcatacta acagatccac
     3901 tttcacattt aagttgtttt tctaatccgc atatgatcaa ttcaaggccg aataagaagg
     3961 ctggctctgc accttggtga tcaaataatt cgatagcttg tcgtaataat ggcggcatac
     4021 tatcagtagt aggtgtttcc ctttcttctt tagcgacttg atgctcttga tcttccaata
     4081 cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata taacgcgttc tctagtgaaa
     4141 aaccttgttg gcataaaaag gctaattgat tttcgagagt ttcatactgt ttttctgtag
     4201 gccgtgtatc tgaatgtact tttgctccat tgcgatgact tagtaaagca catctaaaac
     4261 ttttagcgtt attgcgtaaa aaatcttgcc agctttcccc ttttaaaggg caaaagtgag
     4321 tatggtgcct atctaacatc tcaatggcta aggcgtcgag caaagcccgc ttatttttta
     4381 catgccaata cagtgtaggc tgctctacac caagcttctg ggcgagttta cgggttgtta
     4441 aaccttcgat tccgacctca ttaagcagct ctaatgcgct gttaatcact ttacttttat
     4501 ctaatctaga catatgaatt ggggccgcgg aggctggatc ggtcccggtg tcttctatgg
     4561 aggtcaaaac agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctctg
     4621 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     4681 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     4741 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     4801 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     4861 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     4921 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     4981 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     5041 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     5101 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     5161 attgattact attaataact agtcaataat caatgtcaac atggcggtaa tgttggacat
     5221 gagccaatat aaatgtacat attatgatat ggatacaacg tatgcaatgg gccaagctcc
     5281 tcgagtaatt cgcgccactt ctaaataagc gaatttctta tgatttatga tttttattat
     5341 taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa
     5401 acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata
     5461 gtatgaggtc gctcttattg accacacctc taccggcaga tccgctaggg ataacagggt
     5521 aatatagatc aattcctcga tcgagtttac cactccctat cagtgataga gaaaagtgaa
     5581 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
     5641 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     5701 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     5761 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     5821 gagaaaagtg aaagtcgagc tcggtaccct atggcatgca tgtgctctgt atgtatataa
     5881 aactcttgtt ttcttctttt ctctaaatat tctttcctta tacattaggt cctttgtagc
     5941 ataaattact atacttctat agacacgcaa acacaaatac acacactaaa ttaccggatc
     6001 aattcggggg atcagtatgc agattttcgt caagactttg accggtaaaa ccgcaacatt
     6061 ggaagttgaa tcttccgata ccatcgacaa cgttaagtcg aaaattcaag acaaggaagg
     6121 gatccctggg tctggggctt acccatacga tgttcctgac tatgcgggga atggtatcct
     6181 ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag
     6241 gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc
     6301 ccagactctg aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca
     6361 agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc aagcctggga
     6421 cctctattat catgtgttcc gacgaatctc aaagactagt gaattgcaaa gtaatcagca
     6481 tccctcagca caacgcttat ccgctacagg tgtaaacgca cacggacgtc ctcagggctc
     6541 cacgcagaat gccttgagaa ggactggtag agtcaatgga ggtcaccccg tgactactca
     6601 gatggtggaa acagtgcaaa atctagcccc taacttacat cctgagcaaa ttaggtatag
     6661 tttggaaaac acaggctcag tcgaggaaac agtggaaagg tacttgcgtg gtgatgaatt
     6721 cagctttcca cctgggtttg agccctccag ggcgccaatg ggggccaatg cggctgttga
     6781 taataacgct gctggtggcg gagagtttaa cgatcccaga aagaagaaca tgatttgcgc
     6841 tgagaatcta ctcgataaat tccatgtgga tctcaatgaa gatatgagta acttaagctt
     6901 taaggactta gacattgaag agagaaagag attgttggtt tggcaagcca gaaagaattt
     6961 ggagacaaaa ttgcaaagtg ataaagattt gcaaagtttg ctgactttaa ttaattatac
     7021 tgacatagaa atgaacagat tgggaaagta ggcggccgct agggccctgc aggagggccg
     7081 catcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     7141 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     7201 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     7261 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     7321 tttaatttgc ggccgtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     7381 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     7441 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     7501 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
//
