LOCUS       sequence-420241-        4578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal PCR-based tagging with pCUP1-His7-NUbo in budding yeast 
            with URA3 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4578)
  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 4578)
  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-His7-NUbo
FEATURES             Location/Qualifiers
     source          1..4578
                     /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             1686..1706
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1833..1859
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1869..2147
                     /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(2215..2234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2216..2234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2321..2338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2492..3080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2572..2591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3251..4042,4043..4111))
                     /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     3874..3893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4112..4216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4340..4362)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4462..4481
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4562..4578,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4562..4578,1)
                     /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 tgggccatca tcaccatcat caccatagcg gccagatttt cgtcaagact ttgaccggta
     1741 aaaccgcaac attggaagtt gaatcttccg ataccatcga caacgttaag tcgaaaattc
     1801 aagacaagga agggatccct gggtctgggg cttacccata cgatgttcct gactatgcgg
     1861 ggaatggtat cctctggcat gagatgtggc atgaaggcct ggaagaggca tctcgtttgt
     1921 actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg catgctatga
     1981 tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt
     2041 taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc aaggacctcc
     2101 tccaagcctg ggacctctat tatcatgtgt tccgacgaat ctcaaaggcg acagagaatt
     2161 catcgatgat atcagatcca ctagtggcct atgcggccgc ggatctgccg gtctccctat
     2221 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     2281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4261 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     4321 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4381 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4441 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     4501 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     4561 gatttaggtg acactata
//
