LOCUS       sequence-420594-        5406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo(K48R/K63R)-yeGFP in budding 
            yeast with HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5406)
  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 5406)
  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: pHyg-CUbo(K48R/K63R)-yeGFP
FEATURES             Location/Qualifiers
     source          1..5406
                     /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"
     promoter        1354..1697
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1698..2726
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1698..2726
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2734..2981
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2750..2768)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3043..3062)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3044..3062)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3149..3166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3320..3908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3400..3419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4079..4870,4871..4939))
                     /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     4702..4721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4940..5044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5112..5130
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5168..5190)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5290..5309
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5390..5406,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5390..5406,1)
                     /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 ggtcgacagc 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 tgcgcgccag atctgtttag
     1321 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     1381 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     1441 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     1501 aaaaattacg gctcctcgct gcggacctgc gagcagggaa acgctcccct cacagacgcg
     1561 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     1621 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     1681 tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     1741 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     1801 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     1861 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     1921 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     1981 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     2041 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     2101 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     2161 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     2221 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     2281 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     2341 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     2401 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     2461 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     2521 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     2581 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     2641 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     2701 gcactcgtcc gagggcaaag gaataatctc gagtcatgta attagttatg tcacgcttac
     2761 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     2821 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     2881 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     2941 tgcttgagaa ggttttggga cgctcgaagg ctttaacgag ctcgaattca tcgatgatat
     3001 cagatccact agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat
     3061 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3121 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3181 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3241 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3301 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3361 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3421 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3481 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3541 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3601 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3661 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3721 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3781 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3841 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3901 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3961 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4021 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4081 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4141 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4201 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4261 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4321 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4381 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4441 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4501 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4561 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4621 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4681 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4741 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4801 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4861 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4921 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4981 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5041 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     5101 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     5161 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     5221 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     5281 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     5341 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac
     5401 actata
//
