LOCUS       sequence-77385-e        7168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of wt CRAF S259A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7168)
  TITLE     pBABEpuro-CRAF
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7168)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pBABEpuro-CRAF_S259A
FEATURES             Location/Qualifiers
     source          1..7168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             934..1350
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1318..1334
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1378..1402
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3347..3371)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3407..3427)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3412..3741
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3592..3727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3751..4350
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(3751..3770)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4247..4267
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4449..5042
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5243..5260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5414..6002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5494..5513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6180..6971,6972..7040))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6803..6822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7041..7145)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt
      901 ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt
      961 ttgaccttag atcactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc
     1021 aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg
     1081 ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca
     1141 cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct
     1201 tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttctt
     1261 ccatccgcgc cgtctctccc ccttgaacct cctctttcga ccccgcctca atcctccctt
     1321 tatccagccc tcactccttc tctaggcgcc ggccggatcc cagtgtggtg gtacatcaca
     1381 agtttgtaca aaaaagttgg catggagcac atacagggag cttggaagac gatcagcaat
     1441 ggttttggat tcaaagatgc cgtgtttgat ggctccagct gcatctctcc tacaatagtt
     1501 cagcagtttg gctatcagcg ccgggcatca gatgatggca aactcacaga tccttctaag
     1561 acaagcaaca ctatccgtgt tttcttgccg aacaagcaaa gaacagtggt caatgtgcga
     1621 aatggaatga gcttgcatga ctgccttatg aaagcactca aggtgagggg cctgcaacca
     1681 gagtgctgtg cagtgttcag acttctccac gaacacaaag gtaaaaaagc acgcttagat
     1741 tggaatactg atgctgcgtc tttgattgga gaagaacttc aagtagattt cctggatcat
     1801 gttcccctca caacacacaa ctttgctcgg aagacgttcc tgaagcttgc cttctgtgac
     1861 atctgtcaga aattcctgct caatggattt cgatgtcaga cttgtggcta caaatttcat
     1921 gagcactgta gcaccaaagt acctactatg tgtgtggact ggagtaacat cagacaactc
     1981 ttattgtttc caaattccac tattggtgat agtggagtcc cagcactacc ttctttgact
     2041 atgcgtcgta tgcgagagtc tgtttccagg atgcctgtta gttctcagca cagatattct
     2101 acacctcacg ccttcacctt taacacctcc agtccctcat ctgaaggttc cctctcccag
     2161 aggcagaggt cgacagccac acctaatgtc cacatggtca gcaccaccct gcctgtggac
     2221 agcaggatga ttgaggatgc aattcgaagt cacagcgaat cagcctcacc ttcagccctg
     2281 tccagtagcc ccaacaatct gagcccaaca ggctggtcac agccgaaaac ccccgtgcca
     2341 gcacaaagag agcgggcacc agtatctggg acccaggaga aaaacaaaat taggcctcgt
     2401 ggacagagag attcaagcta ttattgggaa atagaagcca gtgaagtgat gctgtccact
     2461 cggattgggt caggctcttt tggaactgtt tataagggta aatggcacgg agatgttgca
     2521 gtaaagatcc taaaggttgt cgacccaacc ccagagcaat tccaggcctt caggaatgag
     2581 gtggctgttc tgcgcaaaac acggcatgtg aacattctgc ttttcatggg gtacatgaca
     2641 aaggacaacc tggcaattgt gacccagtgg tgcgagggca gcagcctcta caaacacctg
     2701 catgtccagg agaccaagtt tcagatgttc cagctaattg acattgcccg gcagacggct
     2761 cagggaatgg actatttgca tgcaaagaac atcatccata gagacatgaa atccaacaat
     2821 atatttctcc atgaaggctt aacagtgaaa attggagatt ttggtttggc aacagtaaag
     2881 tcacgctgga gtggttctca gcaggttgaa caacctactg gctctgtcct ctggatggcc
     2941 ccagaggtga tccgaatgca ggataacaac ccattcagtt tccagtcgga tgtctactcc
     3001 tatggcatcg tattgtatga actgatgacg ggggagcttc cttattctca catcaacaac
     3061 cgagatcaga tcatcttcat ggtgggccga ggatatgcct ccccagatct tagtaagcta
     3121 tataagaact gccccaaagc aatgaagagg ctggtagctg actgtgtgaa gaaagtaaag
     3181 gaagagaggc ctctttttcc ccagatcctg tcttccattg agctgctcca acactctcta
     3241 ccgaagatca accggagcgc ttccgagcca tccttgcatc gggcagccca cactgaggat
     3301 atcaatgctt gcacgctgac cacgtccccg aggctgcctg tcttctagcc aactttcttg
     3361 tacaaagtgg tgatgtagga attcgccagc acagtggtcg acctgtggaa tgtgtgtcag
     3421 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3481 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3601 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3661 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3721 ggaggcctag gcttttgcaa aaagcttacc atgaccgagt acaagcccac ggtgcgcctc
     3781 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3841 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3901 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3961 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4021 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4081 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4141 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4201 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4261 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagt gcccgaagga ccgcgcgacc
     4321 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     4381 gaaaggagcg cacgacccca tgcatcgata aaataaaaga ttttatttag tctccagaaa
     4441 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     4501 tgcaaggcat ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca
     4561 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     4621 tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     4681 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag
     4741 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     4801 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     4861 cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga
     4921 gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct
     4981 cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca
     5041 tttccgactt gtggtctcgc tgccttggga gggtctcctc tgagtgattg actacccgtc
     5101 agcgggggtc ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt
     5161 tacattaaat ggccatagtt gcattaatga atcggccaac gcgcggggag aggcggtttg
     5221 cgtattggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5281 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5341 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5821 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6121 aatgaagttt gcggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6181 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6241 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6301 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6361 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6421 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6481 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6541 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6601 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6661 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6721 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6781 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6841 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6901 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6961 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7021 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7081 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7141 ccgcgcacat ttccccgaaa agtgccac
//
