LOCUS       sequence-420511-        5549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal PCR-based tagging with CUbo-AID*-yeGFP in budding yeast 
            with HygR selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5549)
  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 5549)
  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-AID*-GFP
FEATURES             Location/Qualifiers
     source          1..5549
                     /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"
     promoter        1497..1840
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1841..2869
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1841..2869
                     /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      2877..3124
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2893..2911)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3186..3205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3187..3205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3292..3309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3463..4051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3543..3562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4222..5013,5014..5082))
                     /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     4845..4864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5083..5187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5255..5273
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5311..5333)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5433..5452
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5533..5549,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5533..5549,1)
                     /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 cagatctgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca
     1501 tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg
     1561 actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt
     1621 ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcggacc tgcgagcagg
     1681 gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata
     1741 taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc
     1801 taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaaaa agcctgaact
     1861 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     1921 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     1981 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     2041 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     2101 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     2161 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     2221 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     2281 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     2341 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     2401 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     2461 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     2521 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     2581 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     2641 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     2701 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     2761 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     2821 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataat ctcgagtcat
     2881 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa
     2941 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     3001 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     3061 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaac
     3121 gagctcgaat tcatcgatga tatcagatcc actagtggcc tatgcggccg cggatctgcc
     3181 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     3241 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3301 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3361 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3421 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3481 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3541 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3601 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3661 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3721 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3781 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3841 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3901 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3961 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4021 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4081 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4141 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4201 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4261 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4321 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4381 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4441 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4501 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4561 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4621 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4681 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4741 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4801 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4861 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4921 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4981 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5041 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5101 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5161 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     5221 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     5281 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5341 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5401 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     5461 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     5521 catacacata cgatttaggt gacactata
//
