LOCUS       sequence-228466-        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 SSAA
FEATURES             Location/Qualifiers
     source          1..7394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        125..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      803..812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             812..859
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(2837..2855)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2838..2855)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2875..2892)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2881..3105
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3151..3579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3238..3257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3448..3469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3588..3608)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3593..3922
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3773..3908
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3835..3854
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3989..4783
                     /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(4043..4062)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4653..4672
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4957..5078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4994..5013)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5048..5067
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5127..5143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5127..5143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5140..5162)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5151..5167
                     /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(5175..5181,5182..5199,5200..5205))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5220..5241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5358..5375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5529..6117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5609..5628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6288..7079,7080..7148))
                     /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     6911..6930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7149..7253)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7328..7347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
       61 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      121 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gcttggtacc gagctcggat ccgccaccat ggactacaag gacgacgatg acaaggacta
      841 caaggacgac gatgacaaga attctcgagt aaccatggag cacatacagg gagcttggaa
      901 gacgatcagc aatggttttg gattcaaaga tgccgtgttt gatggctcca gctgcatctc
      961 tcctacaata gttcagcagt ttggctatca gcgccgggca tcagatgatg gcaaactcac
     1021 agatccttct aagacaagca acactatccg tgttttcttg ccgaacaagc aaagaacagt
     1081 ggtcaatgtg cgaaatggaa tgagcttgca tgactgcctt atgaaagcac tcaaggtgag
     1141 gggcctgcaa ccagagtgct gtgcagtgtt cagacttctc cacgaacaca aaggtaaaaa
     1201 agcacgctta gattggaata ctgatgctgc gtctttgatt ggagaagaac ttcaagtaga
     1261 tttcctggat catgttcccc tcacaacaca caactttgct cggaagacgt tcctgaagct
     1321 tgccttctgt gacatctgtc agaaattcct gctcaatgga tttcgatgtc agacttgtgg
     1381 ctacaaattt catgagcact gtagcaccaa agtacctact atgtgtgtgg actggagtaa
     1441 catcagacaa ctcttattgt ttccaaattc cactattggt gatagtggag tcccagcact
     1501 accttctttg actatgcgtc gtatgcgaga gtctgtttcc aggatgcctg ttagttctca
     1561 gcacagatat tctacacctc acgccttcac ctttaacacc tccagtccct catctgaagg
     1621 ttccctctcc cagaggcaga ggtcgacatc cacacctaat gtccacatgg tcagcaccac
     1681 cctgcctgtg gacagcagga tgattgagga tgcaattcga agtcacagcg aatcagcctc
     1741 accttcagcc ctgtccagta gccccaacaa tctgagccca acaggctggt cacagccgaa
     1801 aacccccgtg ccagcacaaa gagagcgggc accagtatct gggacccagg agaaaaacaa
     1861 aattaggcct cgtggacaga gagattcaag cgctgcttgg gaaatagaag ccagtgaagt
     1921 gatgctgtcc actcggattg ggtcaggctc ttttggaact gtttataagg gtaaatggca
     1981 cggagatgtt gcagtaaaga tcctaaaggt tgtcgaccca accccagagc aattccaggc
     2041 cttcaggaat gaggtggctg ttctgcgcaa aacacggcat gtgaacattc tgcttttcat
     2101 ggggtacatg acaaaggaca acctggcaat tgtgacccag tggtgcgagg gcagcagcct
     2161 ctacaaacac ctgcatgtcc aggagaccaa gtttcagatg ttccagctaa ttgacattgc
     2221 ccggcagacg gctcagggaa tggactattt gcatgcaaag aacatcatcc atagagacat
     2281 gaaatccaac aatatatttc tccatgaagg cttaacagtg aaaattggag attttggttt
     2341 ggcaacagta aagtcacgct ggagtggttc tcagcaggtt gaacaaccta ctggctctgt
     2401 cctctggatg gccccagagg tgatccgaat gcaggataac aacccattca gtttccagtc
     2461 ggatgtctac tcctatggca tcgtattgta tgaactgatg acgggggagc ttccttattc
     2521 tcacatcaac aaccgagatc agatcatctt catggtgggc cgaggatatg cctccccaga
     2581 tcttagtaag ctatataaga actgccccaa agcaatgaag aggctggtag ctgactgtgt
     2641 gaagaaagta aaggaagaga ggcctctttt tccccagatc ctgtcttcca ttgagctgct
     2701 ccaacactct ctaccgaaga tcaaccggag cgcttccgag ccatccttgc atcgggcagc
     2761 ccacactgag gatatcaatg cttgcacgct gaccacgtcc ccgaggctgc ctgtcttcta
     2821 gtctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga
     2881 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2941 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3001 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3061 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3121 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3181 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3241 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3301 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3361 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3421 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3481 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3541 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     3601 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3661 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3721 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3781 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3841 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3901 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3961 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4021 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4081 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4141 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4201 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4261 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     4321 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     4381 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     4441 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     4501 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     4561 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     4621 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4681 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4741 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4801 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4861 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4921 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4981 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5041 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5101 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5161 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5221 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5281 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5341 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5401 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5461 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5521 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5581 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5641 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5701 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5761 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5821 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5881 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5941 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6001 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6061 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6121 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6181 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6241 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6301 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6361 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6421 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6481 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6541 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6601 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6661 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6721 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6781 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6841 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6901 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6961 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7021 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7081 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7141 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7201 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7261 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     7321 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     7381 tgttggaggt cgct
//
