LOCUS       sequence-363392-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector with N-terminal Flag for KBTBD4 R313PRR 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  AUTHORS   Chen Z, Ioris RM, Richardson S, Van Ess AN, Vendrell I, Kessler BM, 
            Buffa FM, Busino L, Clifford SC, Bullock AN, D'Angiolella V
  TITLE     Disease-associated KBTBD4 mutations in medulloblastoma elicit 
            neomorphic ubiquitylation activity to promote CoREST degradation.
  JOURNAL   Cell Death Differ. 2022 Apr 4. pii: 10.1038/s41418-022-00983-4. doi:
            10.1038/s41418-022-00983-4.
  PUBMED    35379950
REFERENCE   2  (bases 1 to 7019)
  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"; journalName: "Cell Death 
            Differ. 2022 Apr 4. pii: 10.1038/s41418-022-00983-4. doi: 
            10.1038/s41418-022-00983-4."
COMMENT     Sequence Label: pcDNA 3.1 KBTBD4 R313PRR
FEATURES             Location/Qualifiers
     source          1..7019
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             898..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKGDYKDDDDKIDYKDDDDK"
     promoter        complement(2572..2590)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2573..2590)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2610..2627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2616..2840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2886..3314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2973..2992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3183..3204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3323..3343)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3328..3657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3508..3643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3570..3589
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3724..4518
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3778..3797)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4388..4407
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4692..4813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4729..4748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4783..4802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4862..4878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4862..4878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4875..4897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4886..4902
                     /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(4910..4916,4917..4934,4935..4940))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4955..4976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5093..5110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5264..5852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5344..5363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6023..6814,6815..6883))
                     /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     6646..6665
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6884..6988)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttatggat
      901 tacaaggatg acgacgataa gggagattac aaggatgacg acgataagat cgattacaag
      961 gatgacgacg ataagggatc catggaatca ccagaggagc ctggagcatc catggatgag
     1021 aactactttg tgaactacac tttcaaagat cggtcacatt caggccgtgt ggctcaaggc
     1081 atcatgaaac tgtgtctaga ggaggagctc tttgctgatg tcaccatttc ggtggaaggc
     1141 cgggagtttc agctccatcg gctggtcctc tcagctcaga gctgcttctt ccgatccatg
     1201 ttcacttcca acctgaagga ggcccacaac cgggtgattg tgctgcagga tgtcagcgag
     1261 tctgttttcc agctcctggt tgattatatc taccatggga ctgtgaaact tcgagctgag
     1321 gagttgcagg aaatttatga ggtgtcagac atgtatcagc tgacatctct ctttgaggaa
     1381 tgctctcggt ttttggcccg cacagtgcaa gtgggaaact gccttcaggt gatgtggctg
     1441 gcagatcggc acagtgatcc tgagctctat acggctgcca agcactgtgc caagacccac
     1501 ctggcccagc tgcagaatac agaggaattt ctccacttgc cccaccgctt actcacagat
     1561 atcatctcgg atggagttcc gtgttctcag aacccaacag aggcaataga agcctggatc
     1621 aactttaata aagaggaaag agaggctttt gcagagtcac tcaggacaag cttgaaggaa
     1681 attggggaga atgtgcacat ttacctgatt gggaaagagt catctcgtac ccactcgttg
     1741 gctgtgtcct tgcactgtgc agaagatgac tccatcagtg taagtggcca aaacagtttg
     1801 tgccaccaga tcactgcggc ctgcaagcat ggtggagact tgtatgtggt gggagggtcc
     1861 atcccacggc cacggcgcat gtggaagtgc aacaatgcca ccgttgactg ggagtggtgt
     1921 gctcctttgc ctcgggaccg gctccagcac accctggtgt ctgtgcccgg gaaagatgcc
     1981 atatattcac tgggtggcaa gacactgcaa gataccctct ccaacgcagt catttattat
     2041 cgcgtaggtg ataatgtgtg gacagagaca actcagctag aggtggctgt gtcaggggct
     2101 gctggtgcca acctcaacgg gatcatctac ttactagggg gggaggagaa tgatctggac
     2161 ttctttacca aaccttcccg actcatccag tgctttgaca cagagacaga caaatgccat
     2221 gtgaagccct atgtgctgcc ctttgcaggc cgcatgcacg cagctgtgca taaagatctg
     2281 gtgttcatcg tggctgaagg ggactccctg gtgtgctaca atcccttgct agacagcttc
     2341 acccggcttt gccttcctga ggcctggagc tctgccccat ccctctggaa gattgccagc
     2401 tgtaacggga gcatctatgt cttccgggac cgatataaaa agggggatgc caacacctac
     2461 aagcttgacc ctgccacttc agccgtaact gtcacaagag gtattaaggt gctgcttacc
     2521 aatttgcagt ttgtgttggc ctaagtcgag catgcatcta gagggcccta ttctatagtg
     2581 tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2641 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2701 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2761 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2821 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2881 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2941 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3001 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3061 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3121 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3181 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3241 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3301 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3361 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3421 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3481 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3541 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3601 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3661 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3721 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3781 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3841 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3901 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3961 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4021 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4081 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4141 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4201 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4261 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4321 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4381 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4441 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4501 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4561 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4621 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4681 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4741 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4801 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4861 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4921 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4981 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5041 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5101 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5161 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5221 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5281 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5341 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5401 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5461 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5521 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5581 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5641 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5701 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5761 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5821 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5881 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5941 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6001 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6061 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6121 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6181 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6241 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6301 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6361 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6421 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6481 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6541 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6601 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6661 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6721 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6781 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6841 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6901 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6961 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
