LOCUS       sequence-420579-        5428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo(K48R/K63R)-yeGFP-His6 in 
            budding yeast with HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5428)
  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 5428)
  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-His6
FEATURES             Location/Qualifiers
     source          1..5428
                     /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..1296
                     /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"
     CDS             1303..1320
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1376..1719
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1720..2748
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1720..2748
                     /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      2756..3003
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2772..2790)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3065..3084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3066..3084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3171..3188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3342..3930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3422..3441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4101..4892,4893..4961))
                     /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     4724..4743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4962..5066)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5134..5152
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5190..5212)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5312..5331
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5412..5428,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5412..5428,1)
                     /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 tacaaaggtt ctcaccacca ccaccaccac
     1321 taagcgcgcc agatctgttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat
     1381 ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga
     1441 ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt
     1501 tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg
     1561 aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat
     1621 aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct
     1681 aggatacagt tctcacatca catccgaaca taaacaacca tgggtaaaaa gcctgaactc
     1741 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     1801 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     1861 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     1921 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     1981 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     2041 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     2101 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     2161 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     2221 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     2281 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     2341 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     2401 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     2461 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     2521 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     2581 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     2641 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     2701 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataatc tcgagtcatg
     2761 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     2821 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     2881 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     2941 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaacg
     3001 agctcgaatt catcgatgat atcagatcca ctagtggcct atgcggccgc ggatctgccg
     3061 gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc
     3121 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3181 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3241 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3301 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3361 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3421 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3481 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3541 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3601 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3661 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3721 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3781 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3841 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3901 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3961 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4021 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4081 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4141 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4201 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4261 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4321 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4381 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4441 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4501 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4561 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4621 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4681 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4741 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4801 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4861 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4921 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4981 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5041 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     5101 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     5161 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     5221 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     5281 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     5341 caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     5401 atacacatac gatttaggtg acactata
//
