LOCUS       sequence-420589-        5555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo-AID*-yeGFP in budding yeast 
            with CaUra3 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5555)
  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 5555)
  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: pCaUra3-CUbo-AID*-GFP
FEATURES             Location/Qualifiers
     source          1..5555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             85..405
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             577..708
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             join(724..726,727..1440)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(724..726,727..1437)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(766..787)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1027..1046)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1374..1395
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1464..1651
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1519..1543)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1726..2069
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      2888..3085
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3192..3211)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3193..3211)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3298..3315)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3469..4057)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3549..3568)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4228..5019,5020..5088))
                     /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     4851..4870
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5089..5193)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5261..5279
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5317..5339)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5439..5458
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5539..5555,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5539..5555,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acgggagcgg agcaggtgct
       61 ggtgctggtg ctggagcaat tctgggagtg caggtggaaa ccatctcccc aggagacggg
      121 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
      181 ggaaagaaat ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
      241 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
      301 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
      361 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaagggtc gaccggtatc
      421 cctccagatc aacaaagatt gatctttgcc ggtaagcagc tagaagacgg tagaacgctg
      481 tctgattaca acattcagaa ggagtccacc ttacatcttg tgctaaggct aagaggtgtt
      541 ggtgcaggcg ccggggctgg tgctggggca ggtaccccta aagatccagc caaacctccg
      601 gccaaggcac aagttgtggg atggccaccg gtgagatcat accggaagaa cgtgatggtt
      661 tcctgccaaa aatcaagcgg tggcccggag gcggcggcgt tcgtgaaggt atcacccggg
      721 aaagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      781 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      841 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      901 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      961 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1021 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1081 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1141 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1201 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1261 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1321 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1381 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1441 aaaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
     1501 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     1561 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg
     1621 aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac agggtaatat
     1681 agatctgttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag
     1741 aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg
     1801 tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc
     1861 acggcgcgaa gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg aaacgctccc
     1921 ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag
     1981 gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt
     2041 tctcacatca catccgaaca taaacaacca tgacagtcaa cactaagacc tatagtgaga
     2101 gagcagaaac tcatgcctca ccagtagcac aacgattatt tcgattaatg gaactgaaga
     2161 aaaccaattt atgtgcatca attgatgttg ataccactaa ggaattcctt gaattaattg
     2221 ataaattggg tccttatgta tgcttaatca agactcatat tgatataatc aatgattttt
     2281 cctatgaatc cactattgaa ccattattag aactttcacg taaacatcaa tttatgattt
     2341 ttgaagatag aaaatttgct gatattggta ataccgtgaa gaaacaatat attggtggag
     2401 tttataaaat tagtagttgg gcagatatta ctaatgctca tggtgtcact gggaatggag
     2461 tagttgaagg attaaaacag ggagctaaag aaaccaccac caaccaagag ccaagagggt
     2521 tattgatgtt agctgaatta tcatcagtgg gatcattagc atatggagaa tattctcaaa
     2581 aaactgttga aattgctaaa tccgataagg aatttgttat tggatttatt gcccaacgtg
     2641 atatgggtgg acaagaagaa ggatttgatt ggcttattat gacacctgga gttggattag
     2701 atgataaagg tgatggatta ggacaacaat atagaactgt tgatgaagtt gttagcactg
     2761 gaactgatat tatcattgtt ggtagaggat tgtttggtaa aggaagagat ccagatattg
     2821 aaggtaaaag gtatagagat gctggttgga atgcttattt gaaaaagact ggccaattat
     2881 aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt
     2941 ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct
     3001 cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg
     3061 tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc
     3121 gaaaacgagc tcgaattcat cgatgatatc agatccacta gtggcctatg cggccgcgga
     3181 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     3241 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3301 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3361 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3421 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3481 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3541 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3601 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3661 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3721 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3781 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3841 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3901 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3961 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4021 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4081 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4141 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4201 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4261 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4321 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4381 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4441 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4501 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4561 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4621 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4681 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4741 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4801 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4861 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4921 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4981 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5041 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5101 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5161 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5221 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5281 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5341 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     5401 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     5461 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     5521 atgtatcata cacatacgat ttaggtgaca ctata
//
