LOCUS       sequence-420240-        4548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal PCR-based tagging with pCUP1-NUbo in budding yeast with 
            URA3 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4548)
  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 4548)
  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-NUbo
FEATURES             Location/Qualifiers
     source          1..4548
                     /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             1803..1829
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1839..2117
                     /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"
     primer_bind     complement(2185..2204)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2186..2204)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2291..2308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2462..3050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2542..2561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3221..4012,4013..4081))
                     /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     3844..3863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4082..4186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4310..4332)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4432..4451
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4532..4548,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4532..4548,1)
                     /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 tgcagatttt cgtcaagact ttgaccggta aaaccgcaac attggaagtt gaatcttccg
     1741 ataccatcga caacgttaag tcgaaaattc aagacaagga agggatccct gggtctgggg
     1801 cttacccata cgatgttcct gactatgcgg ggaatggtat cctctggcat gagatgtggc
     1861 atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt
     1921 ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa
     1981 catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt
     2041 acatgaaatc agggaatgtc aaggacctcc tccaagcctg ggacctctat tatcatgtgt
     2101 tccgacgaat ctcaaaggcg acagagaatt catcgatgat atcagatcca ctagtggcct
     2161 atgcggccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2221 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2281 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2341 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2401 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2461 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2521 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2581 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2641 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2701 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2761 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2821 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2881 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2941 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3001 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3061 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3121 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3181 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3241 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3301 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3361 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3421 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3481 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3541 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3601 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3661 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3721 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3781 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3841 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3901 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3961 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4021 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4081 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4141 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4201 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4261 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4321 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4381 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4441 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     4501 aatacataac cttatgtatc atacacatac gatttaggtg acactata
//
