LOCUS       sequence-420519-        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Copper-inducible expression of the artificial test substrate 
            His6-CUbo(K48R/K63R)-GFP in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  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 6116)
  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: YIp128-CUP1-His6-CUbo(K48R/K63R)-GFP
FEATURES             Location/Qualifiers
     source          1..6116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1995..2400
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2401..3495
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3636..3658)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3758..3777
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3971..3993
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3985..4002
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3986..4002
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4178..4412
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             4462..4479
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(4489..4491,4492..4812)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             4951..5661
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4978..5006)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5601..5621
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5686..5873
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5741..5765)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5897..5913)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5897..5913)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5910..5932)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5921..5937
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5945..5951,5952..5969,5970..5975))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5990..6011
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct
     2041 cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg ctatcgcaca
     2101 gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg acgcatatac
     2161 ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc
     2221 atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa
     2281 tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa
     2341 cttttcttac cttttacatt tcagcaatat atatatatat ttcaaggata taccattcta
     2401 atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc acgttggtca agaaatcaca
     2461 gccgaagcca ttaaggttct taaagctatt tctgatgttc gttccaatgt caagttcgat
     2521 ttcgaaaatc atttaattgg tggtgctgct atcgatgcta caggtgttcc acttccagat
     2581 gaggcgctgg aagcctccaa gaaggctgat gccgttttgt taggtgctgt gggtggtcct
     2641 aaatggggta caggtagtgt tagacctgaa caaggtttac taaaaatccg taaagaactt
     2701 caattgtacg ccaacttaag accatgtaac tttgcatccg actctctttt agacttatct
     2761 ccaatcaagc cacaatttgc taaaggtact gacttcgttg ttgtcagaga attagtggga
     2821 ggtatttact ttggtaagag aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa
     2881 caatacaccg ttccagaagt gcaaagaatc acaagaatgg ccgctttcat ggccctacaa
     2941 catgagccac cattgcctat ttggtccttg gataaagcta atgttttggc ctcttcaaga
     3001 ttatggagaa aaactgtgga ggaaaccatc aagaacgaat ttcctacatt gaaggttcaa
     3061 catcaattga ttgattctgc cgccatgatc ctagttaaga acccaaccca cctaaatggt
     3121 attataatca ccagcaacat gtttggtgat atcatctccg atgaagcctc cgttatccca
     3181 ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt tgccagacaa gaacaccgca
     3241 tttggtttgt acgaaccatg ccacggttct gctccagatt tgccaaagaa taaggtcaac
     3301 cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa
     3361 gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg atgcaggtat cagaactggt
     3421 gatttaggtg gttccaacag taccaccgaa gtcggtgatg ctgtcgccga agaagttaag
     3481 aaaatccttg cttaaaaaga ttctcttttt ttatgatatt tgtacataaa ctttataaat
     3541 gaaattcata atagaaacga cacgaaatta caaaatggaa tatgttcata gggtaggccc
     3601 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3661 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3721 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     3781 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3841 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     3901 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     3961 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattcga gctcccatta
     4021 ccgacatttg ggcgctatac gtgcatatgt tcatgtatgt atctgtattt aaaacacttt
     4081 tgtattattt ttcctcatat atgtgtatag gtttatacgg atgatttaat tattacttca
     4141 ccacccttta tttcaggctg atatcttagc cttgttacta gttagaaaaa gacatttttg
     4201 ctgtcagtca ctgtcaagag attcttttgc tggcatttct tctagaagca aaaagagcga
     4261 tgcgtctttt ccgctgaacc gttccagcaa aaaagactac caacgcaata tggattgtca
     4321 gaatcatata aaagagaagc aaataactcc ttgtcttgta tcaattgcat tataatatct
     4381 tcttgttagt gcaatatcat atagaagtca tcgaaataga tattaagaaa aacaaactgt
     4441 aacggatcca tgggcagcag ccatcatcat catcatcaca gcagcggcat gggagtgcag
     4501 gtggaaacca tctccccagg agacgggcgc accttcccca agcgcggcca gacctgcgtg
     4561 gtgcactaca ccgggatgct tgaagatgga aagaaatttg attcctcccg ggacagaaac
     4621 aagcccttta agtttatgct aggcaagcag gaggtgatcc gaggctggga agaaggggtt
     4681 gcccagatga gtgtgggtca gagagccaaa ctgactatat ctccagatta tgcctatggt
     4741 gccactgggc acccaggcat catcccacca catgccactc tcgtcttcga tgtggagctt
     4801 ctaaaactgg aagggtcgac cggtatccct ccagatcaac aaagattgat ctttgccggt
     4861 cgacagctag aagacggtag aacgctgtct gattacaaca ttcagcgaga gtccacctta
     4921 catcttgtgc taaggctaag aggtgttact agtaaaggag aagaactttt cactggagtt
     4981 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga
     5041 gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga
     5101 aaactacctg ttccatggcc aacactggtc accactttca cttatggtgt tcaatgcttc
     5161 tcgagatacc cagaccatat gaaacggcat gactttttca agagtgccat gcccgaaggt
     5221 tatgtacagg aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa
     5281 gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa
     5341 gaagatggaa acattcttgg acacaaattg gaatacaact ataactcaca caatgtatac
     5401 atcatggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattag acacaacatt
     5461 gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc
     5521 cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc
     5581 aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat
     5641 ggcatggatg aactatacaa ataactgcag ccaagctaat tccgggcgaa tttcttatga
     5701 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     5761 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     5821 ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgcaa
     5881 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5941 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     6001 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6061 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgc
//
