LOCUS       sequence-426725-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of 
            NUbo-E3(63)-mCherry-FLAG in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  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 6873)
  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)-mCherry-FLAG
FEATURES             Location/Qualifiers
     source          1..6873
                     /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..2744
                     /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(2182..2200)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2450..2469
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2754..2777
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2825..2842)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2831..3055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3188..3207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3339..3386
                     /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             3394..4416
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3394..4416
                     /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    4546..4667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4583..4602)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4637..4656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4716..4732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4716..4732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4729..4751)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4740..4756
                     /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(4764..4770,4771..4788,4789..4794))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4809..4830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4947..4964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5118..5706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5198..5217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5877..6668,6669..6737))
                     /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     6500..6519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6738..6842)
                     /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 attaacatgg
     2041 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     2101 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2161 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     2221 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     2281 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2341 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     2401 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2461 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     2521 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     2581 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2641 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     2701 ccgagggccg ccactccacc ggcggcatgg acgagctgta caaattaatt aacgactaca
     2761 aggatgacga cgacaagtaa gcggccgctc gagtctagag ggcccgttta aacccgctga
     2821 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2881 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2941 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3001 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     3061 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     3121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3301 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     3361 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     3421 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3481 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3541 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3601 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3661 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3721 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3781 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3841 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3901 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3961 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     4021 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     4081 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     4141 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     4201 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     4261 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     4321 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4381 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     4441 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4501 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4561 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4621 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4681 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4741 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4801 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4861 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4921 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5041 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5101 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5521 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5701 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5761 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5821 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5881 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5941 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6001 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6061 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6121 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6181 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6241 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6301 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6361 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6421 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6481 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6541 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6601 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6661 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6721 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6841 cgcacatttc cccgaaaagt gccacctgac gtc
//
