LOCUS       sequence-171535-        7521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRAF_S259A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7521)
  AUTHORS   Rose JC, Huang PS, Camp ND, Ye J, Leidal AM, Goreshnik I, Trevillian
            BM, Dickinson MS, Cunningham-Bryant D, Debnath J, Baker D, 
            Wolf-Yadlin A, Maly DJ
  TITLE     A computationally engineered RAS rheostat reveals RAS-ERK signaling 
            dynamics.
  JOURNAL   Nat Chem Biol. 2016 Nov 21. doi: 10.1038/nchembio.2244.
  PUBMED    27870838
REFERENCE   2  (bases 1 to 7521)
  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: "Nat Chem 
            Biol. 2016 Nov 21. doi: 10.1038/nchembio.2244."
COMMENT     Sequence Label: CRAF_S259A_pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..7521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2033..2052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2224..2603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2604..2807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2758..2778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2852..2871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2852..2870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2927..2950
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4932..4949)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4938..5162
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5208..5636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5295..5314)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5505..5526
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5645..5665)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5650..5979
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5830..5965
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5892..5911
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6028..7053
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6028..7053
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    7183..7304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7220..7239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7274..7293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7353..7369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7353..7369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7366..7388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7377..7393
                     /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(7401..7407,7408..7425,7426..7431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7446..7467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat cttattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     2041 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     2101 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2161 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2221 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2281 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2341 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2401 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2461 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2521 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2581 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2641 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2701 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2761 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2821 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2881 ctggctagcg tttaaactta agcttggtac cgagctcgga tccatggatt acaaggatga
     2941 cgatgacaag atggagcaca tacagggagc ttggaagacg atcagcaatg gttttggatt
     3001 caaagatgcc gtgtttgatg gctccagctg catctctcct acaatagttc agcagtttgg
     3061 ctatcagcgc cgggcatcag atgatggcaa actcacagat ccttctaaga caagcaacac
     3121 tatccgtgtt ttcttgccga acaagcaaag aacagtggtc aatgtgcgaa atggaatgag
     3181 cttgcatgac tgccttatga aagcactcaa ggtgaggggc ctgcaaccag agtgctgtgc
     3241 agtgttcaga cttctccacg aacacaaagg taaaaaagca cgcttagatt ggaatactga
     3301 tgctgcgtct ttgattggag aagaacttca agtagatttc ctggatcatg ttcccctcac
     3361 aacacacaac tttgctcgga agacgttcct gaagcttgcc ttctgtgaca tctgtcagaa
     3421 attcctgctc aatggatttc gatgtcagac ttgtggctac aaatttcatg agcactgtag
     3481 caccaaagta cctactatgt gtgtggactg gagtaacatc agacaactct tattgtttcc
     3541 aaattccact attggtgata gtggagtccc agcactacct tctttgacta tgcgtcgtat
     3601 gcgagagtct gtttccagga tgcctgttag ttctcagcac agatattcta cacctcacgc
     3661 cttcaccttt aacacctcca gtccctcatc tgaaggttcc ctctcccaga ggcagaggtc
     3721 gacagccaca cctaatgtcc acatggtcag caccaccctg cctgtggaca gcaggatgat
     3781 tgaggatgca attcgaagtc acagcgaatc agcctcacct tcagccctgt ccagtagccc
     3841 caacaatctg agcccaacag gctggtcaca gccgaaaacc cccgtgccag cacaaagaga
     3901 gcgggcacca gtatctggga cccaggagaa aaacaaaatt aggcctcgtg gacagagaga
     3961 ttcaagctat tattgggaaa tagaagccag tgaagtgatg ctgtccactc ggattgggtc
     4021 aggctctttt ggaactgttt ataagggtaa atggcacgga gatgttgcag taaagatcct
     4081 aaaggttgtc gacccaaccc cagagcaatt ccaggccttc aggaatgagg tggctgttct
     4141 gcgcaaaaca cggcatgtga acattctgct tttcatgggg tacatgacaa aggacaacct
     4201 ggcaattgtg acccagtggt gcgagggcag cagcctctac aaacacctgc atgtccagga
     4261 gaccaagttt cagatgttcc agctaattga cattgcccgg cagacggctc agggaatgga
     4321 ctatttgcat gcaaagaaca tcatccatag agacatgaaa tccaacaata tatttctcca
     4381 tgaaggctta acagtgaaaa ttggagattt tggtttggca acagtaaagt cacgctggag
     4441 tggttctcag caggttgaac aacctactgg ctctgtcctc tggatggccc cagaggtgat
     4501 ccgaatgcag gataacaacc cattcagttt ccagtcggat gtctactcct atggcatcgt
     4561 attgtatgaa ctgatgacgg gggagcttcc ttattctcac atcaacaacc gagatcagat
     4621 catcttcatg gtgggccgag gatatgcctc cccagatctt agtaagctat ataagaactg
     4681 ccccaaagca atgaagaggc tggtagctga ctgtgtgaag aaagtaaagg aagagaggcc
     4741 tctttttccc cagatcctgt cttccattga gctgctccaa cactctctac cgaagatcaa
     4801 ccggagcgct tccgagccat ccttgcatcg ggcagcccac actgaggata tcaatgcttg
     4861 cacgctgacc acgtccccga ggctgcctgt cttctagtga tctagagggc ccgtttaaac
     4921 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4981 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5041 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5101 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     5161 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     5221 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5281 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5341 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5401 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5461 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5521 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5581 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     5641 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     5701 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     5761 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     5821 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5881 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     5941 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     6001 tccattttcg gatctgatca gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc
     6061 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     6121 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     6181 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     6241 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc
     6301 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     6361 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     6421 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     6481 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     6541 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     6601 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     6661 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     6721 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     6781 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     6841 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     6901 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     6961 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     7021 cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc
     7081 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     7141 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     7201 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     7261 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     7321 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     7381 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     7441 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     7501 ggaaacctgt cgtgccagct g
//
