LOCUS       sequence-420492-        8100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-inducible expression of NUbo-E3(63)-VSV in budding 
            yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8100)
  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 8100)
  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: YIp128-TET-NUbo-E3(63)-VSV
FEATURES             Location/Qualifiers
     source          1..8100
                     /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        2035..2440
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2441..3535
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3676..3698)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3798..3817
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(3891..4124)
                     /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(4392..5012)
                     /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     4920..4939
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5092..5116)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(5113..5316)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5142..5162)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5317..5696
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     terminator      5808..5995
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5863..5887)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    join(6053..6071,6072..6094,6095..6113,6114..6136,6137..6155,
                     6156..6178,6179..6197,6198..6220,6221..6239,6240..6262,
                     6263..6281,6282..6304,6305..6323)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             6639..6665
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6675..6953
                     /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             7566..7598
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      7633..7880
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7649..7667)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7921..7937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7921..7937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7934..7956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7945..7961
                     /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(7969..7975,7976..7993,7994..7999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8014..8035
                     /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 ggccaactgt
     2041 gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt
     2101 tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt cgatgactgg
     2161 aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac tgaaaaattg
     2221 ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata gagaagcgtt
     2281 catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa ggacctattg
     2341 ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac cttttacatt
     2401 tcagcaatat atatatatat ttcaaggata taccattcta atgtctgccc ctaagaagat
     2461 cgtcgttttg ccaggtgacc acgttggtca agaaatcaca gccgaagcca ttaaggttct
     2521 taaagctatt tctgatgttc gttccaatgt caagttcgat ttcgaaaatc atttaattgg
     2581 tggtgctgct atcgatgcta caggtgttcc acttccagat gaggcgctgg aagcctccaa
     2641 gaaggctgat gccgttttgt taggtgctgt gggtggtcct aaatggggta caggtagtgt
     2701 tagacctgaa caaggtttac taaaaatccg taaagaactt caattgtacg ccaacttaag
     2761 accatgtaac tttgcatccg actctctttt agacttatct ccaatcaagc cacaatttgc
     2821 taaaggtact gacttcgttg ttgtcagaga attagtggga ggtatttact ttggtaagag
     2881 aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt
     2941 gcaaagaatc acaagaatgg ccgctttcat ggccctacaa catgagccac cattgcctat
     3001 ttggtccttg gataaagcta atgttttggc ctcttcaaga ttatggagaa aaactgtgga
     3061 ggaaaccatc aagaacgaat ttcctacatt gaaggttcaa catcaattga ttgattctgc
     3121 cgccatgatc ctagttaaga acccaaccca cctaaatggt attataatca ccagcaacat
     3181 gtttggtgat atcatctccg atgaagcctc cgttatccca ggttccttgg gtttgttgcc
     3241 atctgcgtcc ttggcctctt tgccagacaa gaacaccgca tttggtttgt acgaaccatg
     3301 ccacggttct gctccagatt tgccaaagaa taaggtcaac cctatcgcca ctatcttgtc
     3361 tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa gaaggtaagg ccattgaaga
     3421 tgcagttaaa aaggttttgg atgcaggtat cagaactggt gatttaggtg gttccaacag
     3481 taccaccgaa gtcggtgatg ctgtcgccga agaagttaag aaaatccttg cttaaaaaga
     3541 ttctcttttt ttatgatatt tgtacataaa ctttataaat gaaattcata atagaaacga
     3601 cacgaaatta caaaatggaa tatgttcata gggtaggccc tttcgtctcg cgcgtttcgg
     3661 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3721 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3781 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     3841 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgcccta cccaccgtac
     3901 tcgtcaattc caagggcatc ggtaaacatc tgctcaaact cgaagtcggc catatccaga
     3961 gcgccgtagg gggcggagtc gtggggggta aatcccggac ccggggaatc cccgtccccc
     4021 aacatgtcca gatcgaaatc gtctagcgcg tcggcatgcg ccatcgccac gtcctcgccg
     4081 tctaagtgga gctcgtcccc caggctgaca tcggtcgggg gggccgtcga cagtctgcgc
     4141 gtgtgtcccg cggggagaaa ggacaggcgc ggagccgcca gccccgcctc ttcgggggcg
     4201 tcgtcgtccg ggagatcgag caggccctcg atggtagacc cgtaattgtt tttcgtacgc
     4261 gcggtaccgc ttacccatct ctccgcatca cctttggtaa aggttctaag cttaggtgag
     4321 aacatccctg cctgaacatg agaaaaaaca gggtactcat actcacttct aagtgacggc
     4381 tgcatactaa cagatccact ttcacattta agttgttttt ctaatccgca tatgatcaat
     4441 tcaaggccga ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt
     4501 cgtaataatg gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga
     4561 tgctcttgat cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct gagtgcatat
     4621 aacgcgttct ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttcgagagtt
     4681 tcatactgtt tttctgtagg ccgtgtatct gaatgtactt ttgctccatt gcgatgactt
     4741 agtaaagcac atctaaaact tttagcgtta ttgcgtaaaa aatcttgcca gctttcccct
     4801 tttaaagggc aaaagtgagt atggtgccta tctaacatct caatggctaa ggcgtcgagc
     4861 aaagcccgct tattttttac atgccaatac agtgtaggct gctctacacc aagcttctgg
     4921 gcgagtttac gggttgttaa accttcgatt ccgacctcat taagcagctc taatgcgctg
     4981 ttaatcactt tacttttatc taatctagac atatgaattg gggccgcgga ggctggatcg
     5041 gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg
     5101 gttcactaaa cgagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     5161 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     5221 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     5281 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     5341 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     5401 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     5461 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     5521 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     5581 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     5641 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaaca
     5701 tggcggtaat gttggacatg agccaatata aatgtacata ttatgatatg gatacaacgt
     5761 atgcaatggg ccaagctcct cgagtaattc gcgccacttc taaataagcg aatttcttat
     5821 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     5881 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     5941 ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagat
     6001 ccgctaggga taacagggta atatagatca attcctcgat cgagtttacc actccctatc
     6061 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
     6121 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
     6181 cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa
     6241 aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagttta
     6301 ccactcccta tcagtgatag agaaaagtga aagtcgagct cggtacccta tggcatgcat
     6361 gtgctctgta tgtatataaa actcttgttt tcttcttttc tctaaatatt ctttccttat
     6421 acattaggtc ctttgtagca taaattacta tacttctata gacacgcaaa cacaaataca
     6481 cacactaaat taccggatca attcggggga tcagtatgca gattttcgtc aagactttga
     6541 ccggtaaaac cgcaacattg gaagttgaat cttccgatac catcgacaac gttaagtcga
     6601 aaattcaaga caaggaaggg atccctgggt ctggggctta cccatacgat gttcctgact
     6661 atgcggggaa tggtatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc
     6721 gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg
     6781 ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc
     6841 gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg
     6901 acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca aagactagtg
     6961 aattcccaaa tctactagta tcctccacca acagagatgt acgactatcg caaacttcgg
     7021 taccaccaaa tgcacttgca ctttctgcac cagatagcaa cactgatgaa gacgcggaaa
     7081 tcctggaaga ttcggtagat caatccggta cggcatgcag gagcgaagaa agttcacaaa
     7141 acgaggagga tcacttctgc cctatttgta actcagatct cacacagttt cctgatgaag
     7201 aagagaccag aaagcacgta gaggattgca tacagagagc tgaaaatgcc caacaacata
     7261 cgaatacgtc agatgcagca gatgactccg taaaagaaag ccctgccttt caaaatagaa
     7321 tgttagtgta caagatatcg cctaatacaa cagataatgc aattaaggag tgcccgattt
     7381 gttttgagaa tatggaaccc ggtgagaaag ttgggagact agaatgcttg tgtgttttcc
     7441 attataagtg tattaagaac tggtttcaca aaagggcgca aatgactgca gcccagaagg
     7501 gaaatggaca tgcttttgtt aaaaggaact tttgtccttt ccatgatgcc gtatttttaa
     7561 ttaattatac tgacatagaa atgaacagat tgggaaagta ggcggccgct agggccctgc
     7621 aggagggccg catcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat
     7681 ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt
     7741 ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt
     7801 acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac
     7861 gctcgaaggc tttaatttgc ggccgtcgac ctgcaggcat gcaagcttgg cgtaatcatg
     7921 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     7981 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     8041 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
//
