LOCUS       sequence-419118-        6102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive/Doxycycline-inducible expression of NUbo-E3(48)-FLAG in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6102)
  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 6102)
  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)-FLAG
FEATURES             Location/Qualifiers
     source          1..6102
                     /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..2006
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2054..2071)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2060..2284
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2417..2436)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2568..2615
                     /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             2623..3645
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2623..3645
                     /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    3775..3896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3812..3831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3866..3885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3945..3961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3945..3961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3958..3980)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3969..3985
                     /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(3993..3999,4000..4017,4018..4023))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4038..4059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4176..4193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4347..4935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4427..4446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5106..5897,5898..5966))
                     /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     5729..5748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5967..6071)
                     /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 acgactacaa ggatgacgac gacaagtaag cggccgctcg agtctagagg gcccgtttaa
     2041 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2101 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2161 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2221 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2281 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2341 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2401 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2461 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2521 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     2581 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     2641 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2701 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2761 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     2821 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     2881 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     2941 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     3001 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     3061 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     3121 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     3181 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     3241 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     3301 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     3361 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     3421 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3481 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3541 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3601 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     3661 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3721 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3781 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3841 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3901 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     3961 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4021 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4081 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4141 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5401 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5701 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
