LOCUS       sequence-418951-        6666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of 
            His8-H2B-CUbo(K63R) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6666)
  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 6666)
  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: pcDNA5-FRT-TO-His8-H2B-CUbo(K63R)
FEATURES             Location/Qualifiers
     source          1..6666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             990..1013
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1014..1391
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             join(1398..1400,1401..1721)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1860..2570
                     /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(1887..1915)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2510..2530
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2618..2635)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2624..2848
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2981..3000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3132..3179
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3187..4209
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3187..4209
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4339..4460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4376..4395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4430..4449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4509..4525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4509..4525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4522..4544)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4533..4549
                     /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(4557..4563,4564..4581,4582..4587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4602..4623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4740..4757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4911..5499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4991..5010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5670..6461,6462..6530))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNYERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6293..6312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6531..6635)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttaccatgc atcaccatca ccatcaccat cacatgccag
     1021 agccagcgaa gtctgctccc gccccgaaaa agggctccaa gaaggcggtg actaaggcgc
     1081 agaagaaaga cggcaagaag cgcaagcgca gccgcaagga gagctattcc atctatgtgt
     1141 acaaggttct gaagcaggtc caccctgaca ccggcatttc gtccaaggcc atgggcatca
     1201 tgaattcgtt tgtgaacgac attttcgagc gcatcgcagg tgaggcttcc cgcctggcgc
     1261 attacaacaa gcgctcgacc atcacctcca gggagatcca gacggccgtg cgcctgctgc
     1321 tgcctgggga gttggccaag cacgccgtgt ccgagggtac taaggccgtc accaagtaca
     1381 ccagcgctaa gggatccatg ggagtgcagg tggaaaccat ctccccagga gacgggcgca
     1441 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa
     1501 agaaatttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg
     1561 aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac
     1621 tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac
     1681 atgccactct cgtcttcgat gtggagcttc taaaactgga agggtcgacc ggtatccctc
     1741 cagatcaaca aagattgatc tttgccggta agcagctaga agacggtaga acgctgtctg
     1801 attacaacat tcagcgagag tccaccttac atcttgtgct aaggctaaga ggtgtttaaa
     1861 gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
     1921 ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac
     1981 ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acactggtca
     2041 ccactttcac ttatggtgtt caatgcttct cgagataccc agaccatatg aaacggcatg
     2101 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
     2161 atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata
     2221 gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg
     2281 aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca
     2341 aagctaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt
     2401 atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt
     2461 ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg
     2521 agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa taagcggccg
     2581 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2641 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2701 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2761 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2821 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2881 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2941 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3001 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3061 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3121 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     3181 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     3241 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     3301 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     3361 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     3421 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     3481 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     3541 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     3601 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     3661 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     3721 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     3781 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     3841 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     3901 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     3961 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     4021 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     4081 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     4141 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     4201 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4261 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4321 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4381 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4441 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     4501 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4561 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4741 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4801 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4861 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4921 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4981 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5041 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5101 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5161 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5221 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5281 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5341 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5401 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5461 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5521 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5581 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5641 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5701 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5761 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5821 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5881 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5941 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6001 tcacgctcgt agtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6061 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6121 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6181 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6241 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6301 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6361 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6421 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6481 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6541 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6601 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6661 gacgtc
//
