LOCUS       sequence-257753-        8571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of Flag tagged BRAF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8571)
  AUTHORS   Noeparast A, Teugels E, Giron P, Verschelden G, De Brakeleer S, 
            Decoster L, De Greve J
  TITLE     Non-V600 BRAF mutations recurrently found in lung cancer predict 
            sensitivity to the combination of Trametinib and Dabrafenib.
  JOURNAL   Oncotarget. 2016 Aug 26;8(36):60094-60108. doi: 
            10.18632/oncotarget.11635. eCollection 2017 Sep 1.
  PUBMED    28947956
REFERENCE   2  (bases 1 to 8571)
  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: 
            "10.18632/oncotarget.11635"; journalName: "Oncotarget"; date: 
            "2016-08-26- 26"; volume: "8"; issue: "36"; pages: "60094-60108"
COMMENT     Sequence Label: p3xFLAG CMV14 / BRAF-D594A
FEATURES             Location/Qualifiers
     source          1..8571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2937..3002
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3013..3635
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3212..3231)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3659..3679)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3677..3974
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4028..4822
                     /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(4082..4101)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4692..4711
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5474..5608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5511..5530)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5565..5584
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5613..5632)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5614..5632)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5646..5662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5646..5662)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5659..5681)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5670..5686
                     /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(5694..5700,5701..5718,5719..5724))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5739..5760
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5877..5894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6048..6636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6128..6147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6807..7598,7599..7667))
                     /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     7430..7449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7668..7772)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7799..8254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7886..7905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8096..8117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8380..8402
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8394..8411
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8395..8411
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agaattaagc ttgcggccgc atggcggcgc tgagcggtgg cggtggtggc ggcgcggagc
      661 cgggccaggc tctgttcaac ggggacatgg agcccgaggc cggcgccggc gccggcgccg
      721 cggcctcttc ggctgcggac cctgccattc cggaggaggt gtggaatatc aaacaaatga
      781 ttaagttgac acaggaacat atagaggccc tattggacaa atttggtggg gagcataatc
      841 caccatcaat atatctggag gcctatgaag aatacaccag caagctagat gcactccaac
      901 aaagagaaca acagttattg gaatctctgg ggaacggaac tgatttttct gtttctagct
      961 ctgcatcaat ggataccgtt acatcttctt cctcttctag cctttcagtg ctaccttcat
     1021 ctctttcagt ttttcaaaat cccacagatg tggcacggag caaccccaag tcaccacaaa
     1081 aacctatcgt tagagtcttc ctgcccaaca aacagaggac agtggtacct gcaaggtgtg
     1141 gagttacagt ccgagacagt ctaaagaaag cactgatgat gagaggtcta atcccagagt
     1201 gctgtgctgt ttacagaatt caggatggag agaagaaacc aattggttgg gacactgata
     1261 tttcctggct tactggagaa gaattgcatg tggaagtgtt ggagaatgtt ccacttacaa
     1321 cacacaactt tgtacgaaaa acgtttttca ccttagcatt ttgtgacttt tgtcgaaagc
     1381 tgcttttcca gggtttccgc tgtcaaacat gtggttataa atttcaccag cgttgtagta
     1441 cagaagttcc actgatgtgt gttaattatg accaacttga tttgctgttt gtctccaagt
     1501 tctttgaaca ccacccaata ccacaggaag aggcgtcctt agcagagact gccctaacat
     1561 ctggatcatc cccttccgca cccgcctcgg actctattgg gccccaaatt ctcaccagtc
     1621 cgtctccttc aaaatccatt ccaattccac agcccttccg accagcagat gaagatcatc
     1681 gaaatcaatt tgggcaacga gaccgatcct catcagctcc caatgtgcat ataaacacaa
     1741 tagaacctgt caatattgat gacttgatta gagaccaagg atttcgtggt gatggaggat
     1801 caaccacagg tttgtctgct accccccctg cctcattacc tggctcacta actaacgtga
     1861 aagccttaca gaaatctcca ggacctcagc gagaaaggaa gtcatcttca tcctcagaag
     1921 acaggaatcg aatgaaaaca cttggtagac gggactcgag tgatgattgg gagattcctg
     1981 atgggcagat tacagtggga caaagaattg gatctggatc atttggaaca gtctacaagg
     2041 gaaagtggca tggtgatgtg gcagtgaaaa tgttgaatgt gacagcacct acacctcagc
     2101 agttacaagc cttcaaaaat gaagtaggag tactcaggaa aacacgacat gtgaatatcc
     2161 tactcttcat gggctattcc acaaagccac aactggctat tgttacccag tggtgtgagg
     2221 gctccagctt gtatcaccat ctccatatca ttgagaccaa atttgagatg atcaaactta
     2281 tagatattgc acgacagact gcacagggca tggattactt acacgccaag tcaatcatcc
     2341 acagagacct caagagtaat aatatatttc ttcatgaaga cctcacagta aaaataggtg
     2401 cttttggtct agctacagtg aaatctcgat ggagtgggtc ccatcagttt gaacagttgt
     2461 ctggatccat tttgtggatg gcaccagaag tcatcagaat gcaagataaa aatccataca
     2521 gctttcagtc agatgtatat gcatttggaa ttgttctgta tgaattgatg actggacagt
     2581 taccttattc aaacatcaac aacagggacc agataatttt tatggtggga cgaggatacc
     2641 tgtctccaga tctcagtaag gtacggagta actgtccaaa agccatgaag agattaatgg
     2701 cagagtgcct caaaaagaaa agagatgaga gaccactctt tccccaaatt ctcgcctcta
     2761 ttgagctgct ggcccgctca ttgccaaaaa ttcaccgcag tgcatcagaa ccctccttga
     2821 atcgggctgg tttccaaaca gaggatttta gtctatatgc ttgtgcttct ccaaaaacac
     2881 ccatccaggc agggggatat ggtgcgtttc ctgtccactc tagaggatcc cgggctgact
     2941 acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca
     3001 agtagtgatc ccgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     3061 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     3121 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     3181 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     3241 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     3301 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     3361 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     3421 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     3481 cctgtccttc tgattttaaa ataactatac cagcaggagg acgtccagac acagcatagg
     3541 ctacctggcc atgcccaacc ggtgggacat ttgagttgct tgcttggcac tgtcctctca
     3601 tgcgttgggt ccactcagta gatgcctgtt gaattgggta cgcggccagc ttggctgtgg
     3661 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3721 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3781 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3841 cccatcccgc ccctaactcc gcctagttcc gcccattctc cgccccatgg ctgactaatt
     3901 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3961 ggaggctttt ttggaggaat tgatcagctt gggatctgat caagagacag gatgaggatc
     4021 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4081 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4141 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4201 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4261 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4321 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4381 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4441 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4501 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4561 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4621 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4681 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4741 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4801 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4861 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4921 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4981 ttcttcgccc accccgggct cgatcccctc gcgagttggt tcagctgctg cctgaggctg
     5041 gacgacctcg cggagttcta ccggcagtgc aaatccgtcg gcatccagga aaccagcagc
     5101 ggctatccgc gcatccatgc ccccgaactg caggagtggg gaggcacgat ggccgctttg
     5161 gtcgacccgg acgggacgct cctgcgcctg atacagaacg aattgcttgc aggcatctca
     5221 tgagtgtgtc ttcccgtttt ccgcctgagg tcactgcgtg gatggagcgc tggcgcctgc
     5281 tgcgcgacgg cgagctgctc accacccact cgccaagctg gaaccgtaaa aaggccgcgt
     5341 tgctggcgtt tttccatagg ctccgccgat cataatcagc cataccacat ttgtagaggt
     5401 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     5461 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     5521 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5581 catcaatgta tcttatcatg tctggatcaa ttccctatag tgagtcgtat taaattcgta
     5641 atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5701 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5761 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5821 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5881 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5941 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6001 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6061 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6721 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6781 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6841 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6901 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6961 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7021 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7081 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7141 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7201 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7261 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7321 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7381 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7441 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7501 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7561 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7621 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7741 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7801 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     7861 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     7921 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     7981 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     8041 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     8101 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     8161 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     8221 gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg ctgcgcaact
     8281 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     8341 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     8401 cgacggccag tgccaagctg atctatacat tgaatcaata ttggcaatta gccatattag
     8461 tcattggtta tatagcataa atcaatattg gctattggcc attgcatacg ttgtatctat
     8521 atcataatat gtacatttat attggctcat gtccaatatg accgccatgt t
//
