LOCUS       sequence-227794-        8154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human Braf.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8154)
  AUTHORS   Takahashi M, Li Y, Dillon TJ, Kariya Y, Stork PJS
  TITLE     Phosphorylation of the C-Raf N-region promotes Raf dimerization.
  JOURNAL   Mol Cell Biol. 2017 Jul 10. pii: MCB.00132-17. doi: 
            10.1128/MCB.00132-17.
  PUBMED    28694330
REFERENCE   2  (bases 1 to 8154)
  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: "Mol Cell 
            Biol. 2017 Jul 10. pii: MCB.00132-17. doi: 10.1128/MCB.00132-17."
COMMENT     Sequence Label: Flag-Braf R188L
FEATURES             Location/Qualifiers
     source          1..8154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        444..647
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     598..618
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     692..711
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        692..710
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             733..780
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(3536..3554)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3537..3554)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3574..3591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3580..3804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3850..4278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3937..3956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4147..4168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4287..4307)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4292..4621
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4472..4607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4534..4553
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4688..5482
                     /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(4742..4761)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5352..5371
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5656..5777
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5693..5712)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5747..5766
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5826..5842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5826..5842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5839..5861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5850..5866
                     /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(5874..5880,5881..5898,5899..5904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5919..5940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6057..6074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6228..6816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6308..6327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6987..7778,7779..7847))
                     /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     7610..7629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7848..7952)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8027..8046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
       61 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      121 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      181 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      361 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      421 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      481 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      541 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      601 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      661 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      721 cttaccacca tggactacaa ggacgacgat gacaaggact acaaggacga cgatgacaag
      781 gcggccgctc gggccccggc tctcggttat aagatggcgg cgctgagcgg tggcggtggt
      841 ggcggcgcgg agccgggcca ggctctgttc aacggggaca tggagcccga ggccggcgcc
      901 ggcgccggcg ccgcggcctc ttcggctgcg gaccctgcca ttccggagga ggtgtggaat
      961 atcaaacaaa tgattaagtt gacacaggaa catatagagg ccctattgga caaatttggt
     1021 ggggagcata atccaccatc aatatatctg gaggcctatg aagaatacac cagcaagcta
     1081 gatgcactcc aacaaagaga acaacagtta ttggaatctc tggggaacgg aactgatttt
     1141 tctgtttcta gctctgcatc aatggatacc gttacatctt cttcctcttc tagcctttca
     1201 gtgctacctt catctctttc agtttttcaa aatcccacag atgtggcacg gagcaacccc
     1261 aagtcaccac aaaaacctat cgttagagtc ttcctgccca acaaacagag gacagtggta
     1321 cctgcaaggt gtggagttac agtccgagac agtctaaaga aagcactgat gatgctgggt
     1381 ctaatcccag agtgctgtgc tgtttacaga attcaggatg gagagaagaa accaattggt
     1441 tgggacactg atatttcctg gcttactgga gaagaattgc atgtggaagt gttggagaat
     1501 gttccactta caacacacaa ctttgtacga aaaacgtttt tcaccttagc attttgtgac
     1561 ttttgtcgaa agctgctttt ccagggtttc cgctgtcaaa catgtggtta taaatttcac
     1621 cagcgttgta gtacagaagt tccactgatg tgtgttaatt atgaccaact tgatttgctg
     1681 tttgtctcca agttctttga acaccaccca ataccacagg aagaggcgtc cttagcagag
     1741 actgccctaa catctggatc atccccttcc gcacccgcct cggactctat tgggccccaa
     1801 attctcacca gtccgtctcc ttcaaaatcc attccaattc cacagccctt ccgaccagca
     1861 gatgaagatc atcgaaatca atttgggcaa cgagaccgat cctcatcagc tcccaatgtg
     1921 catataaaca caatagaacc tgtcaatatt gatgacttga ttagagacca aggatttcgt
     1981 ggtgatggag gatcaaccac aggtttgtct gctacccccc ctgcctcatt acctggctca
     2041 ctaactaacg tgaaagcctt acagaaatct ccaggacctc agcgagaaag gaagtcatct
     2101 tcatcctcag aagacaggaa tcgaatgaaa acacttggta gacgggactc gagtgatgat
     2161 tgggagattc ctgatgggca gattacagtg ggacaaagaa ttggatctgg atcatttgga
     2221 acagtctaca agggaaagtg gcatggtgat gtggcagtga aaatgttgaa tgtgacagca
     2281 cctacacctc agcagttaca agccttcaaa aatgaagtag gagtactcag gaaaacacga
     2341 catgtgaata tcctactctt catgggctat tccacaaagc cacaactggc tattgttacc
     2401 cagtggtgtg agggctccag cttgtatcac catctccata tcattgagac caaatttgag
     2461 atgatcaaac ttatagatat tgcacgacag actgcacagg gcatggatta cttacacgcc
     2521 aagtcaatca tccacagaga cctcaagagt aataatatat ttcttcatga agacctcaca
     2581 gtaaaaatag gtgattttgg tctagctaca gtgaaatctc gatggagtgg gtcccatcag
     2641 tttgaacagt tgtctggatc cattttgtgg atggcaccag aagtcatcag aatgcaagat
     2701 aaaaatccat acagctttca gtcagatgta tatgcatttg ggattgttct gtatgaattg
     2761 atgactggac agttacctta ttcaaacatc aacaacaggg accagataat ttttatggtg
     2821 ggacgaggat acctgtctcc agatctcagt aaggtacgga gtaactgtcc aaaagccatg
     2881 aagagattaa tggcagagtg cctcaaaaag aaaagagatg agagaccact ctttccccaa
     2941 attctcgcct ctattgagct gctggcccgc tcattgccaa aaattcaccg cagtgcatca
     3001 gaaccctcct tgaatcgggc tggtttccaa acagaggatt ttagtctata tgcttgtgct
     3061 tctccaaaaa cacccatcca ggcaggggga tatggtgcgt ttcctgtcca ctgaaacaaa
     3121 tgagtgagag agttcaggag agtagcaaca aaaggaaaat aaatgaacat atgtttgctt
     3181 atatgttaaa ttgaataaaa tactctcttt ttttttaagg tgaaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3301 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3361 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaanaaaaaa aaaaaaaaaa
     3421 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3481 aaaaaaaccc gaattcgtcg acctcgaggg atccgggccc tctagagggc cctattctat
     3541 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     3601 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3661 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3721 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3781 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3841 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3901 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3961 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4021 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4081 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4141 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4201 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4261 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4321 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4381 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4441 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4501 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4561 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4621 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     4681 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4741 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4801 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4861 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4921 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4981 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     5041 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     5101 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5161 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     5221 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5281 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5341 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5401 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5461 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     5521 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5581 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5641 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5701 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5761 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5821 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5881 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6001 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6061 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6121 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6181 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6241 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6301 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6361 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6421 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6481 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6541 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6601 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6661 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6721 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6781 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6841 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6901 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6961 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7021 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7081 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7141 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7201 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7261 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7321 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7381 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7441 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7501 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7561 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7621 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7681 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7741 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7801 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7861 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7921 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7981 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     8041 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     8101 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atga
//
