LOCUS       sequence-227773-        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 R89L
FEATURES             Location/Qualifiers
     source          1..7394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        143..522
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        523..726
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     677..697
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     771..790
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        771..789
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      821..830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             830..877
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(2855..2873)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2856..2873)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2893..2910)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2899..3123
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3169..3597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3256..3275)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3466..3487
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3606..3626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3611..3940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3791..3926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3853..3872
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4007..4801
                     /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(4061..4080)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4671..4690
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4975..5096
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5012..5031)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5066..5085
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5145..5161)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5145..5161)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5158..5180)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5169..5185
                     /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(5193..5199,5200..5217,5218..5223))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5238..5259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5376..5393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5547..6135)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5627..5646)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6306..7097,7098..7166))
                     /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     6929..6948
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7167..7271)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7346..7365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
       61 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      121 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      181 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      241 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      301 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      361 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      421 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      481 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      541 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      601 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      661 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      721 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      781 cactataggg agacccaagc ttggtaccga gctcggatcc gccaccatgg actacaagga
      841 cgacgatgac aaggactaca aggacgacga tgacaagaat tctcgagtaa ccatggagca
      901 catacaggga gcttggaaga cgatcagcaa tggttttgga ttcaaagatg ccgtgtttga
      961 tggctccagc tgcatctctc ctacaatagt tcagcagttt ggctatcagc gccgggcatc
     1021 agatgatggc aaactcacag atccttctaa gacaagcaac actatccgtg ttttcttgcc
     1081 gaacaagcaa agaacagtgg tcaatgtgcg aaatggaatg agcttgcatg actgccttat
     1141 gaaagcactc aaggtgctgg gcctgcaacc agagtgctgt gcagtgttca gacttctcca
     1201 cgaacacaaa ggtaaaaaag cacgcttaga ttggaatact gatgctgcgt ctttgattgg
     1261 agaagaactt caagtagatt tcctggatca tgttcccctc acaacacaca actttgctcg
     1321 gaagacgttc ctgaagcttg ccttctgtga catctgtcag aaattcctgc tcaatggatt
     1381 tcgatgtcag acttgtggct acaaatttca tgagcactgt agcaccaaag tacctactat
     1441 gtgtgtggac tggagtaaca tcagacaact cttattgttt ccaaattcca ctattggtga
     1501 tagtggagtc ccagcactac cttctttgac tatgcgtcgt atgcgagagt ctgtttccag
     1561 gatgcctgtt agttctcagc acagatattc tacacctcac gccttcacct ttaacacctc
     1621 cagtccctca tctgaaggtt ccctctccca gaggcagagg tcgacatcca cacctaatgt
     1681 ccacatggtc agcaccaccc tgcctgtgga cagcaggatg attgaggatg caattcgaag
     1741 tcacagcgaa tcagcctcac cttcagccct gtccagtagc cccaacaatc tgagcccaac
     1801 aggctggtca cagccgaaaa cccccgtgcc agcacaaaga gagcgggcac cagtatctgg
     1861 gacccaggag aaaaacaaaa ttaggcctcg tggacagaga gattcaagct attattggga
     1921 aatagaagcc agtgaagtga tgctgtccac tcggattggg tcaggctctt ttggaactgt
     1981 ttataagggt aaatggcacg gagatgttgc agtaaagatc ctaaaggttg tcgacccaac
     2041 cccagagcaa ttccaggcct tcaggaatga ggtggctgtt ctgcgcaaaa cacggcatgt
     2101 gaacattctg cttttcatgg ggtacatgac aaaggacaac ctggcaattg tgacccagtg
     2161 gtgcgagggc agcagcctct acaaacacct gcatgtccag gagaccaagt ttcagatgtt
     2221 ccagctaatt gacattgccc ggcagacggc tcagggaatg gactatttgc atgcaaagaa
     2281 catcatccat agagacatga aatccaacaa tatatttctc catgaaggct taacagtgaa
     2341 aattggagat tttggtttgg caacagtaaa gtcacgctgg agtggttctc agcaggttga
     2401 acaacctact ggctctgtcc tctggatggc cccagaggtg atccgaatgc aggataacaa
     2461 cccattcagt ttccagtcgg atgtctactc ctatggcatc gtattgtatg aactgatgac
     2521 gggggagctt ccttattctc acatcaacaa ccgagatcag atcatcttca tggtgggccg
     2581 aggatatgcc tccccagatc ttagtaagct atataagaac tgccccaaag caatgaagag
     2641 gctggtagct gactgtgtga agaaagtaaa ggaagagagg cctctttttc cccagatcct
     2701 gtcttccatt gagctgctcc aacactctct accgaagatc aaccggagcg cttccgagcc
     2761 atccttgcat cgggcagccc acactgagga tatcaatgct tgcacgctga ccacgtcccc
     2821 gaggctgcct gtcttctagt ctagagggcc ctattctata gtgtcaccta aatgctagag
     2881 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2941 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3001 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3061 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3121 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3181 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3241 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3301 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3361 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3421 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3481 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3541 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3601 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3661 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3721 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3781 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3841 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3901 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3961 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4021 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4081 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4141 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4201 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4261 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4321 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4381 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4441 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4501 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4561 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4621 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4681 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4741 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4801 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4861 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4921 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4981 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5041 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5101 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5161 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5221 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5281 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5341 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5401 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5461 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5521 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5581 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5641 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5701 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5761 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5821 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5881 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5941 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6001 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6061 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6121 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6181 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6241 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6301 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6361 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6421 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6481 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6541 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6601 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6661 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6721 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6781 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6841 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6901 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6961 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7021 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7081 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7141 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7201 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7261 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     7321 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     7381 atctgctccc tgct
//
