LOCUS       sequence-232219-        7394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human Craf.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7394)
  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 7394)
  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-Craf SSDD
FEATURES             Location/Qualifiers
     source          1..7394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     761..780
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        761..779
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      811..820
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             820..867
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(2845..2863)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2846..2863)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2883..2900)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2889..3113
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3159..3587
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3246..3265)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3456..3477
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3596..3616)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3601..3930
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3781..3916
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3843..3862
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3997..4791
                     /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(4051..4070)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4661..4680
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4965..5086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5002..5021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5056..5075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5135..5151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5135..5151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5148..5170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5159..5175
                     /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(5183..5189,5190..5207,5208..5213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5228..5249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5366..5383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5537..6125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5617..5636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6296..7087,7088..7156))
                     /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     6919..6938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7157..7261)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7336..7355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
       61 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      121 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      721 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      781 agacccaagc ttggtaccga gctcggatcc gccaccatgg actacaagga cgacgatgac
      841 aaggactaca aggacgacga tgacaagaat tctcgagtaa ccatggagca catacaggga
      901 gcttggaaga cgatcagcaa tggttttgga ttcaaagatg ccgtgtttga tggctccagc
      961 tgcatctctc ctacaatagt tcagcagttt ggctatcagc gccgggcatc agatgatggc
     1021 aaactcacag atccttctaa gacaagcaac actatccgtg ttttcttgcc gaacaagcaa
     1081 agaacagtgg tcaatgtgcg aaatggaatg agcttgcatg actgccttat gaaagcactc
     1141 aaggtgaggg gcctgcaacc agagtgctgt gcagtgttca gacttctcca cgaacacaaa
     1201 ggtaaaaaag cacgcttaga ttggaatact gatgctgcgt ctttgattgg agaagaactt
     1261 caagtagatt tcctggatca tgttcccctc acaacacaca actttgctcg gaagacgttc
     1321 ctgaagcttg ccttctgtga catctgtcag aaattcctgc tcaatggatt tcgatgtcag
     1381 acttgtggct acaaatttca tgagcactgt agcaccaaag tacctactat gtgtgtggac
     1441 tggagtaaca tcagacaact cttattgttt ccaaattcca ctattggtga tagtggagtc
     1501 ccagcactac cttctttgac tatgcgtcgt atgcgagagt ctgtttccag gatgcctgtt
     1561 agttctcagc acagatattc tacacctcac gccttcacct ttaacacctc cagtccctca
     1621 tctgaaggtt ccctctccca gaggcagagg tcgacatcca cacctaatgt ccacatggtc
     1681 agcaccaccc tgcctgtgga cagcaggatg attgaggatg caattcgaag tcacagcgaa
     1741 tcagcctcac cttcagccct gtccagtagc cccaacaatc tgagcccaac aggctggtca
     1801 cagccgaaaa cccccgtgcc agcacaaaga gagcgggcac cagtatctgg gacccaggag
     1861 aaaaacaaaa ttaggcctcg tggacagaga gattcaagcg acgactggga aatagaagcc
     1921 agtgaagtga tgctgtccac tcggattggg tcaggctctt ttggaactgt ttataagggt
     1981 aaatggcacg gagatgttgc agtaaagatc ctaaaggttg tcgacccaac cccagagcaa
     2041 ttccaggcct tcaggaatga ggtggctgtt ctgcgcaaaa cacggcatgt gaacattctg
     2101 cttttcatgg ggtacatgac aaaggacaac ctggcaattg tgacccagtg gtgcgagggc
     2161 agcagcctct acaaacacct gcatgtccag gagaccaagt ttcagatgtt ccagctaatt
     2221 gacattgccc ggcagacggc tcagggaatg gactatttgc atgcaaagaa catcatccat
     2281 agagacatga aatccaacaa tatatttctc catgaaggct taacagtgaa aattggagat
     2341 tttggtttgg caacagtaaa gtcacgctgg agtggttctc agcaggttga acaacctact
     2401 ggctctgtcc tctggatggc cccagaggtg atccgaatgc aggataacaa cccattcagt
     2461 ttccagtcgg atgtctactc ctatggcatc gtattgtatg aactgatgac gggggagctt
     2521 ccttattctc acatcaacaa ccgagatcag atcatcttca tggtgggccg aggatatgcc
     2581 tccccagatc ttagtaagct atataagaac tgccccaaag caatgaagag gctggtagct
     2641 gactgtgtga agaaagtaaa ggaagagagg cctctttttc cccagatcct gtcttccatt
     2701 gagctgctcc aacactctct accgaagatc aaccggagcg cttccgagcc atccttgcat
     2761 cgggcagccc acactgagga tatcaatgct tgcacgctga ccacgtcccc gaggctgcct
     2821 gtcttctagt ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2881 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2941 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3001 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3061 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3121 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3181 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3241 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3301 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3361 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3421 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3481 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3541 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3601 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3661 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3721 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3781 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3841 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3901 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3961 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4021 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4081 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4141 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4201 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4261 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4321 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4381 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4441 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4501 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4561 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4621 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4681 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4741 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4801 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4861 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4921 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4981 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5041 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5101 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5161 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5221 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5281 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5341 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5401 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5461 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5521 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5581 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5641 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5701 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5761 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5821 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5881 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5941 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6001 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6061 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6121 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6181 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6241 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6301 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6361 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6421 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6481 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6541 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6601 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6661 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6721 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6781 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6841 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6901 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6961 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7021 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7081 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7141 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7201 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7261 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7321 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7381 tgcttgtgtg ttgg
//
