LOCUS       sequence-420534-        6086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Copper-inducible expression of the artificial test substrate 
            His7-NUbo-GFP in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6086)
  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 6086)
  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-His7-NUbo-GFP
FEATURES             Location/Qualifiers
     source          1..6086
                     /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             4456..4476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             4603..4629
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4639..4917
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             4921..5631
                     /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(4948..4976)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5571..5591
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5656..5843
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5711..5735)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5867..5883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5867..5883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5880..5902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5891..5907
                     /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(5915..5921,5922..5939,5940..5945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5960..5981
                     /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 aacggatcaa tgggccatca tcaccatcat caccatagcg gccagatttt cgtcaagact
     4501 ttgaccggta aaaccgcaac attggaagtt gaatcttccg ataccatcga caacgttaag
     4561 tcgaaaattc aagacaagga agggatccct gggtctgggg cttacccata cgatgttcct
     4621 gactatgcgg ggaatggtat cctctggcat gagatgtggc atgaaggcct ggaagaggca
     4681 tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg
     4741 catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat
     4801 ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc
     4861 aaggacctcc tccaagcctg ggacctctat tatcatgtgt tccgacgaat ctcaaagact
     4921 agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat
     4981 gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa
     5041 cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacactggtc
     5101 accactttca cttatggtgt tcaatgcttc tcgagatacc cagaccatat gaaacggcat
     5161 gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa
     5221 gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat
     5281 agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg
     5341 gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc
     5401 aaagctaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat
     5461 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg
     5521 tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt
     5581 gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa ataactgcag
     5641 ccaagctaat tccgggcgaa tttcttatga tttatgattt ttattattaa ataagttata
     5701 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     5761 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct
     5821 cttattgacc acacctctac cggcatgcaa gcttggcgta atcatggtca tagctgtttc
     5881 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5941 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6001 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6061 ggagaggcgg tttgcgtatt gggcgc
//
