LOCUS       sequence-420591-        5412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo(K63R)-yeGFP in budding yeast 
            with CaUra3 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5412)
  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 5412)
  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(K63R)-yeGFP
FEATURES             Location/Qualifiers
     source          1..5412
                     /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             586..1299
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      1321..1508
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1376..1400)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1583..1926
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      2745..2942
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3049..3068)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3050..3068)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3155..3172)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3326..3914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3406..3425)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4085..4876,4877..4945))
                     /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     4708..4727
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4946..5050)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5118..5136
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5174..5196)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5296..5315
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5396..5412,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5396..5412,1)
                     /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 acattcagcg agagtccacc ttacatcttg tgctaaggct aagaggtgtt
      541 actggatccg gagcaggtgc tggtgctggt gctggagcaa ttctgtctaa aggtgaagaa
      601 ttattcactg gtgttgtccc aattttggtt gaattagatg gtgatgttaa tggtcacaaa
      661 ttttctgtct ccggtgaagg tgaaggtgat gctacttacg gtaaattgac cttaaaattt
      721 atttgtacta ctggtaaatt gccagttcca tggccaacct tagtcactac tttcggttat
      781 ggtgttcaat gttttgcgag atacccagat catatgaaac aacatgactt tttcaagtct
      841 gccatgccag aaggttatgt tcaagaaaga actatttttt tcaaagatga cggtaactac
      901 aagaccagag ctgaagtcaa gtttgaaggt gataccttag ttaatagaat cgaattaaaa
      961 ggtattgatt ttaaagaaga tggtaacatt ttaggtcaca aattggaata caactataac
     1021 tctcacaatg tttacatcat ggctgacaaa caaaagaatg gtatcaaagt taacttcaaa
     1081 attagacaca acattgaaga tggttctgtt caattagctg accattatca acaaaatact
     1141 ccaattggtg atggtccagt cttgttacca gacaaccatt acttatccac tcaatctgcc
     1201 ttatccaaag atccaaacga aaagagagac cacatggtct tgttagaatt tgttactgct
     1261 gctggtatta cccatggtat ggatgaattg tacaaataac tgcgcgccac ttctaaataa
     1321 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     1381 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct
     1441 ttcctgtagg tcaggttgct ttctcaggta tagtatgagg tcgctcttat tgaccacacc
     1501 tctaccggca gatccgctag ggataacagg gtaatataga tctgtttagc ttgcctcgtc
     1561 cccgccgggt cacccggcca gcgacatgga ggcccagaat accctccttg acagtcttga
     1621 cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac atttagccca tacatcccca
     1681 tgtataatca tttgcatcca tacattttga tggccgcacg gcgcgaagca aaaattacgg
     1741 ctcctcgctg cggacctgcg agcagggaaa cgctcccctc acagacgcgt tgaattgtcc
     1801 ccacgccgcg cccctgtaga gaaatataaa aggttaggat ttgccactga ggttcttctt
     1861 tcatatactt ccttttaaaa tcttgctagg atacagttct cacatcacat ccgaacataa
     1921 acaaccatga cagtcaacac taagacctat agtgagagag cagaaactca tgcctcacca
     1981 gtagcacaac gattatttcg attaatggaa ctgaagaaaa ccaatttatg tgcatcaatt
     2041 gatgttgata ccactaagga attccttgaa ttaattgata aattgggtcc ttatgtatgc
     2101 ttaatcaaga ctcatattga tataatcaat gatttttcct atgaatccac tattgaacca
     2161 ttattagaac tttcacgtaa acatcaattt atgatttttg aagatagaaa atttgctgat
     2221 attggtaata ccgtgaagaa acaatatatt ggtggagttt ataaaattag tagttgggca
     2281 gatattacta atgctcatgg tgtcactggg aatggagtag ttgaaggatt aaaacaggga
     2341 gctaaagaaa ccaccaccaa ccaagagcca agagggttat tgatgttagc tgaattatca
     2401 tcagtgggat cattagcata tggagaatat tctcaaaaaa ctgttgaaat tgctaaatcc
     2461 gataaggaat ttgttattgg atttattgcc caacgtgata tgggtggaca agaagaagga
     2521 tttgattggc ttattatgac acctggagtt ggattagatg ataaaggtga tggattagga
     2581 caacaatata gaactgttga tgaagttgtt agcactggaa ctgatattat cattgttggt
     2641 agaggattgt ttggtaaagg aagagatcca gatattgaag gtaaaaggta tagagatgct
     2701 ggttggaatg cttatttgaa aaagactggc caattataat cagtactgac aataaaaaga
     2761 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     2821 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     2881 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     2941 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctcg aattcatcga
     3001 tgatatcaga tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag
     3061 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     3121 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3301 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3361 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3421 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3481 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3541 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3601 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3661 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3721 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3781 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3841 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3901 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3961 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4021 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4081 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4141 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4201 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4261 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4321 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4381 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4441 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4501 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4561 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4621 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4681 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4741 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4801 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4861 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4921 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4981 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5041 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     5101 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     5161 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     5221 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     5281 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat
     5341 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
     5401 ggtgacacta ta
//
