LOCUS       sequence-227786-        7385 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 7385)
  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 7385)
  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 S338A
FEATURES             Location/Qualifiers
     source          1..7385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     738..757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      788..797
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             797..844
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(2813..2831)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2814..2831)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2851..2868)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2857..3081
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3127..3555
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3214..3233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3424..3445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3564..3584)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3569..3898
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3749..3884
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3811..3830
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3965..4759
                     /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(4019..4038)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4629..4648
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4933..5054
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4970..4989)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5024..5043
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5103..5119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5103..5119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5116..5138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5127..5143
                     /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(5151..5157,5158..5175,5176..5181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5196..5217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5334..5351)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5505..6093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5585..5604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6264..7055,7056..7124))
                     /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     6887..6906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7125..7229)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7304..7323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct
      781 cggatccgcc accatggact acaaggacga cgatgacaag gactacaagg acgacgatga
      841 caagaattct atggagcaca tacagggagc ttggaagacg atcagcaatg gttttggatt
      901 caaagatgcc gtgtttgatg gctccagctg catctctcct acaatagttc agcagtttgg
      961 ctatcagcgc cgggcatcag atgatggcaa actcacagat ccttctaaga caagcaacac
     1021 tatccgtgtt ttcttgccga acaagcaaag aacagtggtc aatgtgcgaa atggaatgag
     1081 cttgcatgac tgccttatga aagcactcaa ggtgaggggc ctgcaaccag agtgctgtgc
     1141 agtgttcaga cttctccacg aacacaaagg taaaaaagca cgcttagatt ggaatactga
     1201 tgctgcgtct ttgattggag aagaacttca agtagatttc ctggatcatg ttcccctcac
     1261 aacacacaac tttgctcgga agacgttcct gaagcttgcc ttctgtgaca tctgtcagaa
     1321 attcctgctc aatggatttc gatgtcagac ttgtggctac aaatttcatg agcactgtag
     1381 caccaaagta cctactatgt gtgtggactg gagtaacatc agacaactct tattgtttcc
     1441 aaattccact attggtgata gtggagtccc agcactacct tctttgacta tgcgtcgtat
     1501 gcgagagtct gtttccagga tgcctgttag ttctcagcac agatattcta cacctcacgc
     1561 cttcaccttt aacacctcca gtccctcatc tgaaggttcc ctctcccaga ggcagaggtc
     1621 gacatccaca cctaatgtcc acatggtcag caccaccctg cctgtggaca gcaggatgat
     1681 tgaggatgca attcgaagtc acagcgaatc agcctcacct tcagccctgt ccagtagccc
     1741 caacaatctg agcccaacag gctggtcaca gccgaaaacc cccgtgccag cacaaagaga
     1801 gcgggcacca gtatctggga cccaggagaa aaacaaaatt aggcctcgtg gacagagaga
     1861 tgcaagctat tattgggaaa tagaagccag tgaagtgatg ctgtccactc ggattgggtc
     1921 aggctctttt ggaactgttt ataagggtaa atggcacgga gatgttgcag taaagatcct
     1981 aaaggttgtc gacccaaccc cagagcaatt ccaggccttc aggaatgagg tggctgttct
     2041 gcgcaaaaca cggcatgtga acattctgct tttcatgggg tacatgacaa aggacaacct
     2101 ggcaattgtg acccagtggt gcgagggcag cagcctctac aaacacctgc atgtccagga
     2161 gaccaagttt cagatgttcc agctaattga cattgcccgg cagacggctc agggaatgga
     2221 ctatttgcat gcaaagaaca tcatccatag agacatgaaa tccaacaata tatttctcca
     2281 tgaaggctta acagtgaaaa ttggagattt tggtttggca acagtaaagt cacgctggag
     2341 tggttctcag caggttgaac aacctactgg ctctgtcctc tggatggccc cagaggtgat
     2401 ccgaatgcag gataacaacc cattcagttt ccagtcggat gtctactcct atggcatcgt
     2461 attgtatgaa ctgatgacgg gggagcttcc ttattctcac atcaacaacc gagatcagat
     2521 catcttcatg gtgggccgag gatatgcctc cccagatctt agtaagctat ataagaactg
     2581 ccccaaagca atgaagaggc tggtagctga ctgtgtgaag aaagtaaagg aagagaggcc
     2641 tctttttccc cagatcctgt cttccattga gctgctccaa cactctctac cgaagatcaa
     2701 ccggagcgct tccgagccat ccttgcatcg ggcagcccac actgaggata tcaatgcttg
     2761 cacgctgacc acgtccccga ggctgcctgt cttctagtct agagggccct attctatagt
     2821 gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     2881 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2941 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3001 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3061 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3121 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3181 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3241 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3301 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3361 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3421 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3481 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3541 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3601 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3661 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3721 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3781 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3841 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3901 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3961 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4021 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4081 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4141 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4201 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4261 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4321 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4381 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4441 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4501 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4561 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4621 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4681 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4741 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4801 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4861 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4921 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4981 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5041 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5281 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5941 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6121 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6181 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6241 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6301 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6361 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6421 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6481 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6541 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6601 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6661 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6721 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6781 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6841 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6901 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6961 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7021 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7081 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7141 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7201 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7261 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     7321 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     7381 cgagc
//
