LOCUS       sequence-215437-        9154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mutant RASA2 in mamalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9154)
  AUTHORS   Arafeh R, Qutob N, Emmanuel R, Keren-Paz A, Madore J, Elkahloun A, 
            Wilmott JS, Gartner JJ, Di Pizio A, Winograd-Katz S, Sindiri S, 
            Rotkopf R, Dutton-Regester K, Johansson P, Pritchard AL, Waddell N, 
            Hill VK, Lin JC, Hevroni Y, Rosenberg SA, Khan J, Ben-Dor S, Niv MY,
            Ulitsky I, Mann GJ, Scolyer RA, Hayward NK, Samuels Y
  TITLE     Recurrent inactivating RASA2 mutations in melanoma.
  JOURNAL   Nat Genet. 2015 Dec;47(12):1408-10. doi: 10.1038/ng.3427. Epub 2015 
            Oct 26.
  PUBMED    26502337
REFERENCE   2  (bases 1 to 9154)
  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"; doi: "10.1038/ng.3427"; 
            journalName: "Nat Genet"; date: "2015-12"; volume: "47"; issue: 
            "12"; pages: "1408-10"
COMMENT     Sequence Label: pCDF1-S400F_RASA2-EF1-Puro
FEATURES             Location/Qualifiers
     source          1..9154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        133..336
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1479..1682
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1679..1703
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1766..1775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1775..1798
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        4387..4598
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             4611..4879
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             4906..5505
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4906..4925)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5402..5422
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5512..6100
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5565..5585)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5983..5994)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6438..6559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6475..6494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6529..6548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6578..6713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6751..6767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6751..6767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6764..6786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6775..6791
                     /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(6799..6805,6806..6823,6824..6829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6844..6865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6982..6999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7153..7741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7233..7252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7912..8703,8704..8772))
                     /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     8535..8554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8773..8877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8945..8963
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9001..9023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9123..9142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
       61 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      121 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      181 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      241 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      301 aggcgtgtac ggtgggaggt ctatataagc agagcttgtg aaacttcgag gagtctcttt
      361 gttgaggact tttgagttct cccttgaggc tcccacagat acaataaata tttgagattg
      421 aaccctgtcg agtatctgtg taatcttttt tacctgtgag gtctcggaat ccgggccgag
      481 aacttcgcag ttggcgcccg aacagggact tgattgagag tgattgagga agtgaagcta
      541 gagcaataga aagctgttaa gcagaactcc tgctgaccta aatagggaag cagtagcaga
      601 cgctgctaac agtgagtatc tctagtgaag cagactcgag ctcataatca agtcattgtt
      661 taaaggccca gataaattac atctggtgac tcttcgcgga ccttcaagcc aggagattcg
      721 ccgagggaca gtcaacaagg taggagagat tctacagcaa catggggaat ggacaggggc
      781 gagattggaa aatggccatt aagagatgta gtaatgttgc tgtaggagta ggggggaaga
      841 gtaaaaaatt tggagaaggg aatttcagat gggccattag aatggctaat gtatctacag
      901 gacgagaacc tggtgatata ccagagactt tagatcaact aaggttggtt atttgcgatt
      961 tacaagaaag aagagaaaaa tttggatcta gcaaagaaat tgatatggca attcctgcat
     1021 tgaggagaaa tggtaggcaa tgtggcatgt ctgaaaaaga ggaggaatga tgaagtatct
     1081 cagacttatt ttataaggga gatactgtgc tgagttcttc cctttgagga aggtatgtca
     1141 tatcctagac atagtctcaa ttttaaaaga agaggtagga taggagggat ggccccttat
     1201 gaattattag cacaacaaga atccttaaga atacaagatt atttttctgc aataccacaa
     1261 aaattgcaag cacagtggat ttattataaa gatcaaaaag ataagaaatg gaaaggacca
     1321 atgagagtag aatactgggg acagggatca gtattattaa aggatgaaga gaagggatat
     1381 tttcttataa tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt
     1441 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1501 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1561 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1621 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1681 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     1741 gaagattcta gagccaccaa ctagagccac catggattac aaggatgacg acgataaggc
     1801 ggcggcggcg cctgctgctg cggcggcttc ttccgaggcg ccagcggcga gtgcgactgc
     1861 agagcccgag gccggggacc aggacagtcg cgaggttcga gtgttgcaga gcctgcgggg
     1921 caagatctgt gaagcaaaaa atttattgcc atatcttgga ccccacaaaa tgagagattg
     1981 tttctgtacc ataaatttgg accaggaaga agtttatcgt acccaagttg tggaaaaatc
     2041 tttaagccca tttttcagtg aagaatttta ctttgagatt ccaagaactt tccagtattt
     2101 gtctttctat gtttatgata agaatgtttt acaaagagat ctccgtatag gaaaagtagc
     2161 catcaaaaaa gaagacttgt gtaatcacag tggcaaagaa acttggtttt cattacagcc
     2221 tgttgactcc aattcagagg ttcagggtaa agttcacctt gaattaaaac tgaatgaact
     2281 gataacggag aatggaactg tatgccagca gcttgttgta cacatcaagg catgccatgg
     2341 gttgcctctc ataaatggcc aaagctgtga cccttatgca acagtttctc tagtgggccc
     2401 ttctaggaat gaccaaaaga agacaaaagt aaagaagaaa acaagcaatc cgcagtttaa
     2461 tgaaatcttt tattttgaag taaccagatc cagtagttac accagaaagt cccagttcca
     2521 ggtagaagag gaggacattg aaaagctaga aatcaggatc gacttgtgga acaatggaaa
     2581 cctagtccaa gatgttttcc taggtgagat taaggttcct gtgaacgtat taagaactga
     2641 ttcctctcat caagcctggt acttgctaca gccaagagac aatggaaaca agtcatccaa
     2701 aactgatgac ctggggtctc ttcgattaaa tatatgttat acagaagact acgtgcttcc
     2761 ttcagagtac tatggtcctt tgaaaacttt gctgctaaaa tcaccagatg ttcaaccaat
     2821 atctgcctca gctgcttaca ttttgagtga aatatgtcga gataaaaatg atgctgtttt
     2881 gccccttgta cgactgctgc tgcaccatga taaacttgtt ccttttgcca ctgctgtggc
     2941 tgaattagac ttgaaggata cacaagatgc aaacacaatt tttagaggaa atttcctggc
     3001 tacccgatgt ctggatgaga tgatgaaaat agtgggaggg cactacctga aagtaacatt
     3061 aaaacctatt cttgatgaga tatgtgactc ctcaaaatcc tgtgaaatcg atcctattaa
     3121 attgaaagag ggagataatg tagaaaataa taaggagaat ctgcgctact atgtagacaa
     3181 gttattcaat acaattgtaa aatcaagtat gagctgcccc actgtaatgt gtgatatctt
     3241 ttattctcta aggcagatgg ctactcagag atttcctaat gaccctcatg ttcagtattc
     3301 tgcagtgagc agctttgtat ttcttcgttt ctttgctgta gccgtagtat cacctcatac
     3361 ttttcatttg cgacctcatc atccagatgc acagacaatt agaacattaa ctctcatctc
     3421 aaaaactata caaactttgg gaagctgggg gagtctgtcc aaaagcaagt caagtttcaa
     3481 agagacattc atgtgtgaat ttttcaaaat gtttcaagaa gaaggatata ttatagcagt
     3541 taaaaagttc ttggatgaaa tttcatctac tgaaactaaa gagtccagtg gtacgagtga
     3601 gcctgtgcac ctgaaagaag gtgagatgta taaaagagct caaggaagaa ctcggattgg
     3661 aaaaaagaat tttaagaaac gatggttctg cttaacaagc agagagctca cctaccacaa
     3721 acagccaggc aaagatgcaa tctacacaat cccagtaaaa aacattcttg ctgtggaaaa
     3781 actggaagag agctctttca acaagaaaaa tatgttccaa gtaatacata cggagaaacc
     3841 actctatgtc caggcaaata actgtgtaga agctaatgaa tggatagacg tactctgcag
     3901 ggtgagccga tgcaatcaaa acaggctcag tttttatcat ccctctgtgt atctaaacgg
     3961 aaattggctc tgctgtcagg agactggtga aaacactctc ggctgcaagc catgtactgc
     4021 aggtgtccct gcagacatcc aaatagatat tgatgaagac agagaaacag aaagaattta
     4081 ttcccttttt accctcagtt tacttaagct gcagaagatg gaagaggctt gtggaactat
     4141 tgcagtctat caaggaccac agaaagagcc tgatgattat tctaactttg taatcgagga
     4201 ttctgtaaca acctttaaga caattcagca aataaaaagc ataattgaga agctggatga
     4261 acctcatgaa aaatatagga agaaaagatc cagtagtgca aaatatggga gcaaggaaaa
     4321 tccaattgtt gggaaagcat cttaggcggc cgcgaaggat ctgcgatcgc tccggtgccc
     4381 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     4441 attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     4501 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     4561 acgttctttt tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca
     4621 tctctccttc acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg
     4681 ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa
     4741 agctcaggtc gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc
     4801 ggctctccac gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg
     4861 ttctgcgccg ttacagatcc aagctgtgac cggcgcctac gctagatgac cgagtacaag
     4921 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     4981 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     5041 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     5101 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     5161 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     5221 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     5281 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     5341 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     5401 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     5461 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagtcga caatcaacct
     5521 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     5581 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     5641 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     5701 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     5761 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     5821 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     5881 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     5941 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     6001 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     6061 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgggtaccg atgacagagt
     6121 tagaagatcg cttcaggaag ctatttggca cgacttctac aacgggagac agcacagtag
     6181 attctgaaga tgaacctcct aaaaaagaaa aaagggtgga ctgggatgag tattggaacc
     6241 ctgaaatcga tagcttccag tgctttgtga aacttcgagg agtctctttg ttgaggactt
     6301 ttgagttctc ccttgaggct cccacagata caataaatat ttgagattga accctgtcga
     6361 gtatctgtgt aatctttttt acctgtgagg tctcggaatc cgggccgaga acttcgcagc
     6421 gagctcattg taccgcgaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6481 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6541 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccag
     6601 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     6661 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagactt
     6721 ttgcagagac ggcccaaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6781 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6841 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6901 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6961 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     7021 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     7081 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     7141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     7201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     7261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     7321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     7381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     7441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     7501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     7561 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     7621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7801 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     8041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     8101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     8161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     8221 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8521 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     8821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     8881 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     8941 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     9001 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     9061 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     9121 tgcggcatca gagcagattg tactgagagt gcac
//
