LOCUS       sequence-420540-        5076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo(K63R)-9myc in budding yeast 
            with HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5076)
  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 5076)
  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(K63R)-9myc
FEATURES             Location/Qualifiers
     source          1..5076
                     /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             571..600
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             607..636
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             643..672
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             691..720
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             727..756
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             763..792
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             811..840
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             847..876
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             883..912
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        1024..1367
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1368..2396
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1368..2396
                     /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      2404..2651
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2420..2438)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2713..2732)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2714..2732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2819..2836)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2990..3578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3070..3089)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3749..4540,4541..4609))
                     /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     4372..4391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4610..4714)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4782..4800
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4838..4860)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4960..4979
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5060..5076,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5060..5076,1)
                     /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 actggatcat ccggttctgc tgctagtggt gaacaaaagt tgatttctga agaagatttg
      601 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtgaacaaaa attaatctca
      661 gaagaagact tgaacggatc ctctagaggt gaacaaaagt tgatttctga agaagatttg
      721 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtgaacaaaa attaatctca
      781 gaagaagact tgaacggatc ctctagaggt gaacaaaagt tgatttctga agaagatttg
      841 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtgaacaaaa attaatctca
      901 gaagaagact tgaacggatc cactagctaa aaagtggaac gatcattcaa gagatccccg
      961 ggttaattaa ggcgcgccag atctgtttag cttgcctcgt ccccgccggg tcacccggcc
     1021 agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca
     1081 tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc
     1141 atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcggacctgc
     1201 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
     1261 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
     1321 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaaaaagc
     1381 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     1441 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     1501 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     1561 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     1621 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     1681 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     1741 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     1801 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     1861 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     1921 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     1981 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     2041 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     2101 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     2161 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     2221 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     2281 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     2341 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataatctc
     2401 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     2461 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     2521 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     2581 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     2641 ctttaacgag ctcgaattca tcgatgatat cagatccact agtggcctat gcggccgcgg
     2701 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     2761 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2821 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2881 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2941 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3001 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3061 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3121 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3181 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3241 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3301 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3361 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3421 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3481 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3541 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3601 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3661 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3721 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3781 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3841 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3901 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3961 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4021 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4081 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4141 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4201 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4261 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4321 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4381 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4441 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4501 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4561 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4621 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4681 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4741 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4801 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4861 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4921 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4981 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     5041 tatgtatcat acacatacga tttaggtgac actata
//
