LOCUS       sequence-419005-        6156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of NUbo-E3(63)-FLAG
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6156)
  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 6156)
  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-NUbo-E3(63)-FLAG
FEATURES             Location/Qualifiers
     source          1..6156
                     /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             1107..1133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1107..1133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2037..2060
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2108..2125)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2114..2338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2471..2490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2622..2669
                     /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             2677..3699
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2677..3699
                     /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    3829..3950
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3866..3885)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3920..3939
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3999..4015)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3999..4015)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4012..4034)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4023..4039
                     /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(4047..4053,4054..4071,4072..4077))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4092..4113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4230..4247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4401..4989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4481..4500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5160..5951,5952..6020))
                     /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     5783..5802
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6021..6125)
                     /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 cttatgcaga tcttcgtgaa gaccctgacc ggcaagaccg
     1021 ccaccctgga ggtggagagc agcgacacca tcgacaacgt gaagagcaag atccaggaca
     1081 aggagggcat ccccggcagc ggcgcctacc cctacgacgt gcccgactac gccggcaacg
     1141 gcatcctgtg gcacgagatg tggcacgagg gcctggagga ggccagccgc ctgtacttcg
     1201 gcgagcgcaa cgtgaagggc atgttcgagg tgctggagcc cctgcacgcc atgatggagc
     1261 gcggccccca gaccctgaag gagaccagct tcaaccaggc ctacggccgc gacctgatgg
     1321 aggcccagga gtggtgccgc aagtacatga agagcggcaa cgtgaaggac ctgctgcagg
     1381 cctgggacct gtactaccac gtgttccgcc gcatcagcaa gactagtact agtgagctga
     1441 acctgctggt gagcagcacc aaccgcgacg tgcgcctgag ccagaccagc gtgcccccca
     1501 acgccctggc cctgagcgcc cccgacagca acaccgacga ggacgccgag atcctggagg
     1561 acagcgtgga ccagagcggc accgcctgcc gcagcgagga gagcagccag aacgaggagg
     1621 accacttctg ccccatctgc aacagcgacc tgacccagtt ccccgacgag gaggagaccc
     1681 gcaagcacgt ggaggactgc atccagcgcg ccgagaacgc ccagcagcac accaacacca
     1741 gcgacgccgc cgacgacagc gtgaaggaga gccccgcctt ccagaaccgc atgctggtgt
     1801 acaagatcag ccccaacacc accgacaacg ccatcaagga gtgccccatc tgcttcgaga
     1861 acatggagcc cggcgagaag gtgggccgcc tggagtgcct gtgcgtgttc cactacaagt
     1921 gcatcaagaa ctggttccac aagcgcgccc agatgaccgc cgcccagaag ggcaacggcc
     1981 acgccttcgt gaagcgcaac ttctgcccct tccacgacgc cgtgttctta attaacgact
     2041 acaaggatga cgacgacaag taagcggccg ctcgagtcta gagggcccgt ttaaacccgc
     2101 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2161 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2221 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2281 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2341 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2401 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2461 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2521 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2581 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     2641 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     2701 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     2761 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     2821 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     2881 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     2941 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3001 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3061 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3121 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3181 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3241 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3301 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3361 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3421 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     3481 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     3541 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     3601 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     3661 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     3721 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3781 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3841 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3901 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3961 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4021 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4081 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4141 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4201 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4261 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4321 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4381 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4441 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4501 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4561 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4621 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4681 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4741 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4801 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4861 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4921 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4981 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5041 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5101 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5161 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5221 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5281 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5341 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5401 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5461 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5521 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5581 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5641 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5701 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5761 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5821 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5881 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5941 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6001 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6061 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6121 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
