LOCUS       sequence-227776-        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 R401H
FEATURES             Location/Qualifiers
     source          1..7394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..711
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     662..682
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     756..775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        756..774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      806..815
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             815..862
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(2840..2858)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2841..2858)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2878..2895)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2884..3108
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3154..3582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3241..3260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3451..3472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3591..3611)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3596..3925
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3776..3911
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3838..3857
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3992..4786
                     /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(4046..4065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4656..4675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4960..5081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4997..5016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5051..5070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5130..5146)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5130..5146)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5143..5165)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5154..5170
                     /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(5178..5184,5185..5202,5203..5208))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5223..5244
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5361..5378)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5532..6120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5612..5631)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6291..7082,7083..7151))
                     /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     6914..6933
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7152..7256)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7331..7350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
      721 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
      781 caagcttggt accgagctcg gatccgccac catggactac aaggacgacg atgacaagga
      841 ctacaaggac gacgatgaca agaattctcg agtaaccatg gagcacatac agggagcttg
      901 gaagacgatc agcaatggtt ttggattcaa agatgccgtg tttgatggct ccagctgcat
      961 ctctcctaca atagttcagc agtttggcta tcagcgccgg gcatcagatg atggcaaact
     1021 cacagatcct tctaagacaa gcaacactat ccgtgttttc ttgccgaaca agcaaagaac
     1081 agtggtcaat gtgcgaaatg gaatgagctt gcatgactgc cttatgaaag cactcaaggt
     1141 gaggggcctg caaccagagt gctgtgcagt gttcagactt ctccacgaac acaaaggtaa
     1201 aaaagcacgc ttagattgga atactgatgc tgcgtctttg attggagaag aacttcaagt
     1261 agatttcctg gatcatgttc ccctcacaac acacaacttt gctcggaaga cgttcctgaa
     1321 gcttgccttc tgtgacatct gtcagaaatt cctgctcaat ggatttcgat gtcagacttg
     1381 tggctacaaa tttcatgagc actgtagcac caaagtacct actatgtgtg tggactggag
     1441 taacatcaga caactcttat tgtttccaaa ttccactatt ggtgatagtg gagtcccagc
     1501 actaccttct ttgactatgc gtcgtatgcg agagtctgtt tccaggatgc ctgttagttc
     1561 tcagcacaga tattctacac ctcacgcctt cacctttaac acctccagtc cctcatctga
     1621 aggttccctc tcccagaggc agaggtcgac atccacacct aatgtccaca tggtcagcac
     1681 caccctgcct gtggacagca ggatgattga ggatgcaatt cgaagtcaca gcgaatcagc
     1741 ctcaccttca gccctgtcca gtagccccaa caatctgagc ccaacaggct ggtcacagcc
     1801 gaaaaccccc gtgccagcac aaagagagcg ggcaccagta tctgggaccc aggagaaaaa
     1861 caaaattagg cctcgtggac agagagattc aagctattat tgggaaatag aagccagtga
     1921 agtgatgctg tccactcgga ttgggtcagg ctcttttgga actgtttata agggtaaatg
     1981 gcacggagat gttgcagtaa agatcctaaa ggttgtcgac ccaaccccag agcaattcca
     2041 ggccttcagg aatgaggtgg ctgttctgcg caaaacacat catgtgaaca ttctgctttt
     2101 catggggtac atgacaaagg acaacctggc aattgtgacc cagtggtgcg agggcagcag
     2161 cctctacaaa cacctgcatg tccaggagac caagtttcag atgttccagc taattgacat
     2221 tgcccggcag acggctcagg gaatggacta tttgcatgca aagaacatca tccatagaga
     2281 catgaaatcc aacaatatat ttctccatga aggcttaaca gtgaaaattg gagattttgg
     2341 tttggcaaca gtaaagtcac gctggagtgg ttctcagcag gttgaacaac ctactggctc
     2401 tgtcctctgg atggccccag aggtgatccg aatgcaggat aacaacccat tcagtttcca
     2461 gtcggatgtc tactcctatg gcatcgtatt gtatgaactg atgacggggg agcttcctta
     2521 ttctcacatc aacaaccgag atcagatcat cttcatggtg ggccgaggat atgcctcccc
     2581 agatcttagt aagctatata agaactgccc caaagcaatg aagaggctgg tagctgactg
     2641 tgtgaagaaa gtaaaggaag agaggcctct ttttccccag atcctgtctt ccattgagct
     2701 gctccaacac tctctaccga agatcaaccg gagcgcttcc gagccatcct tgcatcgggc
     2761 agcccacact gaggatatca atgcttgcac gctgaccacg tccccgaggc tgcctgtctt
     2821 ctagtctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct
     2881 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2941 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3001 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3061 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3121 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3181 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3241 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3301 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3361 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3421 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3481 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3541 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3601 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3661 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3721 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3781 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3841 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3901 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3961 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4021 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4081 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4141 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4201 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4261 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4321 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4381 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4441 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4501 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4561 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4621 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4681 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4741 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4801 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4861 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4921 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4981 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5041 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     5101 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5161 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5221 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5281 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5341 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5401 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5461 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5521 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5581 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5641 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5701 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5761 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5821 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5881 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5941 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6001 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6061 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6121 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6181 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6241 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6301 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6361 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6421 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6481 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6541 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6601 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6661 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6721 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6781 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6841 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6901 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6961 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7021 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7081 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7141 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7201 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7261 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     7321 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7381 gtgtgttgga ggtc
//
