LOCUS       sequence-418954-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive or doxycycline-inducible expression of 
            His6-NUbo-H2B-His8 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  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 5952)
  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-His6-NUbo-H2B-His8
FEATURES             Location/Qualifiers
     source          1..5952
                     /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             987..1004
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1134..1160
                     /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             1134..1160
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1455..1832
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             1833..1856
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1904..1921)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1910..2134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2267..2286)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2418..2465
                     /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             2473..3495
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2473..3495
                     /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    3625..3746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3662..3681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3716..3735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3795..3811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3795..3811)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3808..3830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3819..3835
                     /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(3843..3849,3850..3867,3868..3873))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3888..3909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4026..4043)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4197..4785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4277..4296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4956..5747,5748..5816))
                     /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     5579..5598
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5817..5921)
                     /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 cttatgcatc accatcacca tcacaagctt gggcagatct
     1021 tcgtgaagac cctgaccggc aagaccgcca ccctggaggt ggagagcagc gacaccatcg
     1081 acaacgtgaa gagcaagatc caggacaagg agggcatccc cggcagcggc gcctacccct
     1141 acgacgtgcc cgactacgcc ggcaacggca tcctgtggca cgagatgtgg cacgagggcc
     1201 tggaggaggc cagccgcctg tacttcggcg agcgcaacgt gaagggcatg ttcgaggtgc
     1261 tggagcccct gcacgccatg atggagcgcg gcccccagac cctgaaggag accagcttca
     1321 accaggccta cggccgcgac ctgatggagg cccaggagtg gtgccgcaag tacatgaaga
     1381 gcggcaacgt gaaggacctg ctgcaggcct gggacctgta ctaccacgtg ttccgccgca
     1441 tcagcaagac tagtatgcca gagccagcga agtctgctcc cgccccgaaa aagggctcca
     1501 agaaggcggt gactaaggcg cagaagaaag acggcaagaa gcgcaagcgc agccgcaagg
     1561 agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac accggcattt
     1621 cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag cgcatcgcag
     1681 gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc agggagatcc
     1741 agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg tccgagggta
     1801 ctaaggccgt caccaagtac accagcgcta agcatcacca tcaccatcac catcactaag
     1861 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     1921 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1981 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2041 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2101 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2161 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2221 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2281 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2341 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2401 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     2461 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2521 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2581 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2641 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2701 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2761 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2821 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2881 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2941 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     3001 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3061 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3121 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3181 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3241 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3301 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3361 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3421 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3481 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3541 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3601 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3661 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3721 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3781 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3841 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3901 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3961 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4021 gcttcctcgc tcactgactc gctgcgcgcg gtcgttcggc tgcggcgagc ggtatcagct
     4081 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4141 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4201 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4261 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4321 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4381 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4441 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4501 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4561 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4621 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4681 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4741 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4801 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4861 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4921 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4981 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5041 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5101 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5161 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5221 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5281 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5341 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5401 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5461 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5521 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5581 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5641 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5701 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5761 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5821 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5881 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5941 ccacctgacg tc
//
