LOCUS       sequence-363387-        7016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector with N-terminal Flag for KBTBD4 R312RG 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7016)
  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 7016)
  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 R312RG
FEATURES             Location/Qualifiers
     source          1..7016
                     /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(2569..2587)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2570..2587)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2607..2624)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2613..2837
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2883..3311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2970..2989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3180..3201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3320..3340)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3325..3654
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3505..3640
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3567..3586
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3721..4515
                     /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(3775..3794)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4385..4404
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4689..4810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4726..4745)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4780..4799
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4859..4875)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4859..4875)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4872..4894)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4883..4899
                     /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(4907..4913,4914..4931,4932..4937))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4952..4973
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5090..5107)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5261..5849)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5341..5360)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6020..6811,6812..6880))
                     /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     6643..6662
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6881..6985)
                     /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 atcccacggg gacgcatgtg gaagtgcaac aatgccaccg ttgactggga gtggtgtgct
     1921 cctttgcctc gggaccggct ccagcacacc ctggtgtctg tgcccgggaa agatgccata
     1981 tattcactgg gtggcaagac actgcaagat accctctcca acgcagtcat ttattatcgc
     2041 gtaggtgata atgtgtggac agagacaact cagctagagg tggctgtgtc aggggctgct
     2101 ggtgccaacc tcaacgggat catctactta ctaggggggg aggagaatga tctggacttc
     2161 tttaccaaac cttcccgact catccagtgc tttgacacag agacagacaa atgccatgtg
     2221 aagccctatg tgctgccctt tgcaggccgc atgcacgcag ctgtgcataa agatctggtg
     2281 ttcatcgtgg ctgaagggga ctccctggtg tgctacaatc ccttgctaga cagcttcacc
     2341 cggctttgcc ttcctgaggc ctggagctct gccccatccc tctggaagat tgccagctgt
     2401 aacgggagca tctatgtctt ccgggaccga tataaaaagg gggatgccaa cacctacaag
     2461 cttgaccctg ccacttcagc cgtaactgtc acaagaggta ttaaggtgct gcttaccaat
     2521 ttgcagtttg tgttggccta agtcgagcat gcatctagag ggccctattc tatagtgtca
     2581 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2641 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2701 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2761 tggggtgggg caggacagca agtgggagga ttgggaagac aatagcaggc atgctgggga
     2821 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2881 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2941 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3001 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3061 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3121 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3181 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3241 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3301 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3361 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3421 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3481 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3541 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3601 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3661 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3721 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3781 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3841 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3901 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3961 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4021 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4081 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4141 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4201 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4261 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4321 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4381 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4441 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4501 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4561 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4621 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4681 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4741 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4801 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4861 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4921 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4981 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5041 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5101 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5161 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5221 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5281 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5341 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5401 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5461 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5521 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5581 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5641 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5701 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5761 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5821 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5881 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5941 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6001 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6061 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6121 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6181 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6241 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6301 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6361 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6421 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6481 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6541 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6601 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6661 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6721 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6781 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6841 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6901 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6961 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
