LOCUS       sequence-420251-        4569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal PCR-based tagging with pCUP1-CUbo(K63R) in budding yeast 
            with URA3 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4569)
  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 4569)
  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: pUra3-pCUP1-CUbo(K63R)
FEATURES             Location/Qualifiers
     source          1..4569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        93..308
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             309..1112
                     /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        1383..1617
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             join(1680..1682,1683..2003)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     primer_bind     complement(2206..2225)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2207..2225)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2312..2329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2483..3071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2563..2582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3242..4033,4034..4102))
                     /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     3865..3884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4103..4207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4331..4353)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4453..4472
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4553..4569,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4553..4569,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 aggcgcgcca gatcttatca cgaggccagc ttttcaattc atcatttttt ttttattctt
      121 ttttttgatt tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag
      181 aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa
      241 catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacatgc aggaaacgaa
      301 gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg
      361 ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg
      421 ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac
      481 taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa
      541 aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg
      601 gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca
      661 ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag
      721 aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct
      781 ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt
      841 ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt
      901 tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata
      961 gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat
     1021 ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag
     1081 catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta
     1141 tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc gccctttcgt
     1201 gagctctagt aagccgatcc cattaccgac atttgggcgc tatacgtgca tatgttcatg
     1261 tatgtatctg tatttaaaac acttttgtat tatttttcct catatatgtg tataggttta
     1321 tacggatgat ttaattatta cttcaccacc ctttatttcg ggctgatatc ttagccttgt
     1381 tactagttag aaaaagacat ttttgctgtc agtcactgtc aagagattct tttgctggca
     1441 tttcttctag aagcaaaaag agcgatgcgt cttttccgct gaaccgttcc agcaaaaaag
     1501 actaccaacg caatatggat tgtcagaatc atataaaaga gaagcaaata actccttgtc
     1561 ttgtatcaat tgcattataa tatcttcttg ttagtgcaat atcatataga agtcatcgaa
     1621 atagatatta agaaaaacaa actgtacaat caatcaatca atcatcacat aaatccggaa
     1681 tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc aagcgcggcc
     1741 agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaattt gattcctccc
     1801 gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc cgaggctggg
     1861 aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt
     1921 atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact ctcgtcttcg
     1981 atgtggagct tctaaaactg gaagggtcga ccggtatccc tccagatcaa caaagattga
     2041 tctttgccgg taagcagcta gaagacggta gaacgctgtc tgattacaac attcagcgag
     2101 agtccacctt acatcttgtg ctaaggctaa gaggtgttgc gacagagaat tcatcgatga
     2161 tatcagatcc actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg
     2221 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     2281 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2401 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2461 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2521 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2581 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2641 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2701 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2761 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2821 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2881 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2941 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3001 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3061 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3121 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3181 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3241 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3301 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3361 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3421 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3481 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3541 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3601 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     3661 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3721 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3781 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3841 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3901 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3961 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4021 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4081 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4141 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4201 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4261 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     4321 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4381 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     4441 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     4501 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
     4561 gacactata
//
