LOCUS       sequence-363391-        7013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector with N-terminal 3xFlag for KBTBD4 WT 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7013)
  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 7013)
  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 WT
FEATURES             Location/Qualifiers
     source          1..7013
                     /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(2566..2584)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2567..2584)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2604..2621)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2610..2834
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2880..3308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2967..2986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3177..3198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3317..3337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3322..3651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3502..3637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3564..3583
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3718..4512
                     /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(3772..3791)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4382..4401
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4686..4807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4723..4742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4777..4796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4856..4872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4856..4872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4869..4891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4880..4896
                     /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(4904..4910,4911..4928,4929..4934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4949..4970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5087..5104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5258..5846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5338..5357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6017..6808,6809..6877))
                     /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     6640..6659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6878..6982)
                     /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 gcatgtggaa gtgcaacaat gccaccgttg actgggagtg gtgtgctcct
     1921 ttgcctcggg accggctcca gcacaccctg gtgtctgtgc ccgggaaaga tgccatatat
     1981 tcactgggtg gcaagacact gcaagatacc ctctccaacg cagtcattta ttatcgcgta
     2041 ggtgataatg tgtggacaga gacaactcag ctagaggtgg ctgtgtcagg ggctgctggt
     2101 gccaacctca acgggatcat ctacttacta gggggggagg agaatgatct ggacttcttt
     2161 accaaacctt cccgactcat ccagtgcttt gacacagaga cagacaaatg ccatgtgaag
     2221 ccctatgtgc tgccctttgc aggccgcatg cacgcagctg tgcataaaga tctggtgttc
     2281 atcgtggctg aaggggactc cctggtgtgc tacaatccct tgctagacag cttcacccgg
     2341 ctttgccttc ctgaggcctg gagctctgcc ccatccctct ggaagattgc cagctgtaac
     2401 gggagcatct atgtcttccg ggaccgatat aaaaaggggg atgccaacac ctacaagctt
     2461 gaccctgcca cttcagccgt aactgtcaca agaggtatta aggtgctgct taccaatttg
     2521 cagtttgtgt tggcctaagt cgagcatgca tctagagggc cctattctat agtgtcacct
     2581 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2641 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2701 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2761 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2821 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2881 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2941 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3001 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3061 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3121 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3181 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3241 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3301 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3361 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3421 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3481 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3541 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3601 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3661 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3721 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3781 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3841 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3901 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3961 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4021 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4081 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4141 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4201 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4261 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4321 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4381 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4441 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4501 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4561 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4621 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4681 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4801 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
