LOCUS       sequence-420238-        6474 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive expression of NUbo-E3(63)-mCherry-VSV in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  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 6474)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-ADH-NUbo-E3(63)-mCherry-VSV
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2000..2215
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2216..3019
                     /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(3137..3159)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3259..3278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3472..3494
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3486..3503
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3487..3503
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3508..4211
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             4345..4371
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4381..4659
                     /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             5269..5976
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5414..5432)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5682..5701
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5986..6018
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     terminator      6044..6231
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6099..6123)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6255..6271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6255..6271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6268..6290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6279..6295
                     /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(6303..6309,6310..6327,6328..6333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6348..6369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccagcttt tcaattcatc attttttttt tattcttttt tttgatttcg
     2041 gtttccttga aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga
     2101 gcacagactt agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc
     2161 agtattctta acccaactgc acagaacaaa aacatgcagg aaacgaagat aaatcatgtc
     2221 gaaagctaca tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt
     2281 taatatcatg cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa
     2341 ggaattactg gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt
     2401 ggatatcttg actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc
     2461 caagtacaat tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa
     2521 attgcagtac tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca
     2581 cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa
     2641 ggaacctaga ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg
     2701 agaatatact aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt
     2761 tattgctcaa agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc
     2821 cggtgtgggt ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga
     2881 tgtggtctct acaggatctg acattattat tgttggaaga ggactatttg caaagggaag
     2941 ggatgctaag gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag
     3001 atgcggccag caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca
     3061 aattagagct tcaatttaat tatatcagtt attacccgcc ctttcgtctc gcgcgtttcg
     3121 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3181 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3241 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     3301 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     3361 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     3421 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     3481 gacgttgtaa aacgacggcc agtgaattcc ttttgttgtt tccgggtgta caatatggac
     3541 ttcctctttt ctggcaacca aacccataca tcgggattcc tataatacct tcgttggtct
     3601 ccctaacatg taggtggcgg aggggagata tacaatagaa cagataccag acaagacata
     3661 atgggctaaa caagactaca ccaattacac tgcctcattg atggtggtac ataacgaact
     3721 aatactgtag ccctagactt gatagccatc atcatatcga agtttcacta ccctttttcc
     3781 atttgccatc tattgaagta ataataggcg catgcaactt cttttctttt tttttctttt
     3841 ctctctcccc cgttgttgtc tcaccatatc cgcaatgaca aaaaaatgat ggaagacact
     3901 aaaggaaaaa attaacgaca aagacagcac caacagatgt cgttgttcca gagctgatga
     3961 ggggtatctc gaagcacacg aaactttttc cttccttcat tcacgcacac tactctctaa
     4021 tgagcaacgg tatacggcct tccttccagt tacttgaatt tgaaataaaa aaaagtttgc
     4081 tgtcttgcta tcaagtataa atagacctgc aattattaat cttttgtttc ctcgtcattg
     4141 ttctcgttcc ctttcttcct tgtttctttt tctgcacaat atttcaagct ataccaagca
     4201 tacaatcaac tccggatcag tatgcagatt ttcgtcaaga ctttgaccgg taaaaccgca
     4261 acattggaag ttgaatcttc cgataccatc gacaacgtta agtcgaaaat tcaagacaag
     4321 gaagggatcc ctgggtctgg ggcttaccca tacgatgttc ctgactatgc ggggaatggt
     4381 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg
     4441 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg
     4501 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag
     4561 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc
     4621 tgggacctct attatcatgt gttccgacga atctcaaaga ctagcgaatt gaatctacta
     4681 gtatcctcca ccaacagaga tgtacgacta tcgcaaactt cggtaccacc aaatgcactt
     4741 gcactttctg caccagatag caacactgat gaagacgcgg aaatcctgga agattcggta
     4801 gatcaatccg gtacggcatg caggagcgaa gaaagttcac aaaacgagga ggatcacttc
     4861 tgccctattt gtaactcaga tctcacacag tttcctgatg aagaagagac cagaaagcac
     4921 gtagaggatt gcatacagag agctgaaaat gcccaacaac atacgaatac gtcagatgca
     4981 gcagatgact ccgtaaaaga aagccctgcc tttcaaaata gaatgttagt gtacaagata
     5041 tcgcctaata caacagataa tgcaattaag gagtgcccga tttgttttga gaatatggaa
     5101 cccggtgaga aagttgggag actagaatgc ttgtgtgttt tccattataa gtgtattaag
     5161 aactggtttc acaaaagggc gcaaatgact gcagcccaga agggaaatgg acatgctttt
     5221 gttaaaagga acttttgtcc tttccatgat gccgtatttt taattaacat ggtgagcaag
     5281 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     5341 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     5401 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     5461 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     5521 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     5581 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     5641 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     5701 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     5761 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     5821 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     5881 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     5941 cgccactcca ccggcggcat ggacgagctg tacaaattaa ttaattatac tgacatagaa
     6001 atgaacagat tgggaaagta gcctgcagcc aagctaattc cgggcgaatt tcttatgatt
     6061 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     6121 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     6181 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgcaagc
     6241 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6301 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6361 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6421 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgc
//
