LOCUS       sequence-426723-        6819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of 
            NUbo-E3(48)-mCherry-FLAG in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6819)
  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 6819)
  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(48)-mCherry-FLAG
FEATURES             Location/Qualifiers
     source          1..6819
                     /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             1983..2690
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2128..2146)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2396..2415
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2700..2723
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2771..2788)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2777..3001
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3134..3153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3285..3332
                     /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             3340..4362
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3340..4362
                     /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    4492..4613
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4529..4548)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4583..4602
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4662..4678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4662..4678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4675..4697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4686..4702
                     /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(4710..4716,4717..4734,4735..4740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4755..4776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4893..4910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5064..5652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5144..5163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5823..6614,6615..6683))
                     /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     6446..6465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6684..6788)
                     /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 gactagtgag ctgcagagca
     1441 accagcaccc aagcgcacag cgtctgagcg caaccggtgt gaacgcacac ggtcgtccac
     1501 agggtagcac ccagaacgct ctgcgccgta ccggccgtgt gaacggcggc cacccagtga
     1561 ccacccagat ggtggagacc gtgcagaacc tggcccccaa cctgcacccc gagcagatcc
     1621 gctacagcct ggagaacacc ggcagcgtgg aggagaccgt ggagcgctac ctgcgcggcg
     1681 acgagttcag cttcccacca ggtttcgagc caagccgcgc tccaatgggt gccaacgctg
     1741 ctgtggacaa caacgccgcc ggtggtggtg agttcaacga cccccgcaag aagaacatga
     1801 tctgcgccga gaacctgctg gacaagttcc acgtggacct gaacgaggac atgagcaacc
     1861 tgagcttcaa ggacctggac atcgaggagc gcaagcgcct gctggtgtgg caggcccgca
     1921 agaacctgga gaccaagctg cagagcgaca aggacctgca gagcctgctg accttaatta
     1981 acatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2041 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2101 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2161 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2221 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2281 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2341 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2401 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2461 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2521 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2581 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2641 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaaa ttaattaacg
     2701 actacaagga tgacgacgac aagtaagcgg ccgctcgagt ctagagggcc cgtttaaacc
     2761 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2821 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2881 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2941 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3001 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3061 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     3301 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     3361 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3421 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     3481 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     3541 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     3601 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3661 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     3721 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3781 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3841 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     3901 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     3961 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     4021 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     4081 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     4141 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4201 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     4261 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     4321 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     4381 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4441 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4501 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4561 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4621 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4681 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4741 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4801 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4861 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4921 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4981 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5041 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5101 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5161 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5221 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5281 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5341 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5401 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5461 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5521 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5581 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5641 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5701 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5761 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5821 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5881 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5941 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6001 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6061 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6121 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6181 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6241 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6301 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6361 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6421 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6481 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6541 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6601 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6661 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6721 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6781 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
