LOCUS       sequence-221961-        7536 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 7536)
  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 7536)
  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-R310_RASA2-EF1-Puro
FEATURES             Location/Qualifiers
     source          1..7536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1143..1346
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1343..1367
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1417..1426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1426..1449
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2418..2629
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2642..2910
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2937..3536
                     /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(2937..2956)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3433..3453
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3543..4131
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3596..3616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4014..4025)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4468..4589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4559..4578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4629..4764
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4797..4813)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4797..4813)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4810..4832)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4821..4837
                     /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(4845..4851,4852..4869,4870..4875))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4890..4911
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5028..5045)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5199..5787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5279..5298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5958..6749,6750..6818))
                     /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     6581..6600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6819..6923)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6991..7009
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7047..7069)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7169..7188
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7333..7536
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 tgtgaaactt cgaggagtct ctttgttgag gacttttgag ttctcccttg aggctcccac
       61 agatacaata aatatttgag attgaaccct gtcgagtatc tgtgtaatct tttttacctg
      121 tgaggtctcg gaatccgggc cgagaacttc gcagttggcg cccgaacagg gacttgattg
      181 agagtgattg aggaagtgaa gctagagcaa tagaaagctg ttaagcagaa ctcctgctga
      241 cctaaatagg gaagcagtag cagacgctgc taacagtgag tatctctagt gaagcagact
      301 cgagctcata atcaagtcat tgtttaaagg cccagataaa ttacatctgg tgactcttcg
      361 cggaccttca agccaggaga ttcgccgagg gacagtcaac aaggtaggag agattctaca
      421 gcaacatggg gaatggacag gggcgagatt ggaaaatggc cattaagaga tgtagtaatg
      481 ttgctgtagg agtagggggg aagagtaaaa aatttggaga agggaatttc agatgggcca
      541 ttagaatggc taatgtatct acaggacgag aacctggtga tataccagag actttagatc
      601 aactaaggtt ggttatttgc gatttacaag aaagaagaga aaaatttgga tctagcaaag
      661 aaattgatat ggcaattcct gcattgagga gaaatggtag gcaatgtggc atgtctgaaa
      721 aagaggagga atgatgaagt atctcagact tattttataa gggagatact gtgctgagtt
      781 cttccctttg aggaaggtat gtcatatcct agacatagtc tcaattttaa aagaagaggt
      841 aggataggag ggatggcccc ttatgaatta ttagcacaac aagaatcctt aagaatacaa
      901 gattattttt ctgcaatacc acaaaaattg caagcacagt ggatttatta taaagatcaa
      961 aaagataaga aatggaaagg accaatgaga gtagaatact ggggacaggg atcagtatta
     1021 ttaaaggatg aagagaaggg atattttctt ataatcgata ctagtattat gcccagtaca
     1081 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1141 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     1201 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1261 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1321 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     1381 atccacgctg ttttgacctc catagaagat tctagagcca ccatggatta caaggatgac
     1441 gacgataagg cggcggcggc gcctgctgct gcggcggctt cttccgaggc gccagcggcg
     1501 agtgcgactg cagagcccga ggccggggac caggacagtc gcgaggttcg agtgttgcag
     1561 agcctgcggg gcaagatctg tgaagcaaaa aatttattgc catatcttgg accccacaaa
     1621 atgagagatt gtttctgtac cataaatttg gaccaggaag aagtttatcg tacccaagtt
     1681 gtggaaaaat ctttaagccc atttttcagt gaagaatttt actttgagat tccaagaact
     1741 ttccagtatt tgtctttcta tgtttatgat aagaatgttt tacaaagaga tctccgtata
     1801 ggaaaagtag ccatcaaaaa agaagacttg tgtaatcaca gtggcaaaga aacttggttt
     1861 tcattacagc ctgttgactc caattcagag gttcagggta aagttcacct tgaattaaaa
     1921 ctgaatgaac tgataacgga gaatggaact gtatgccagc agcttgttgt acacatcaag
     1981 gcatgccatg ggttgcctct cataaatggc caaagctgtg acccttatgc aacagtttct
     2041 ctagtgggcc cttctaggaa tgaccaaaag aagacaaaag taaagaagaa aacaagcaat
     2101 ccgcagttta atgaaatctt ttattttgag gtaaccagat ccagtagtta caccagaaag
     2161 tcccagttcc aggtagaaga ggaggacatt gaaaagctag aaatcaggat cgacttgtgg
     2221 aacaatggaa acctagtcca agatgttttc ctaggtgaga ttaaggttcc tgtgaacgta
     2281 ttaagaactg attcctctca tcaagcctgg tacttgctac agccaagaga caatggaaac
     2341 aagtcatcca aaactgatga cctggggtct ctttgagcgg ccgcgaagga tctgcgatcg
     2401 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2461 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2521 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2581 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
     2641 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
     2701 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
     2761 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
     2821 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
     2881 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta cgctagatga
     2941 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     3001 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     3061 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3121 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3181 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3241 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3301 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3361 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3421 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3481 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgagtcg
     3541 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3601 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3661 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3721 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3781 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3841 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3901 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3961 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     4021 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     4081 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggtaccg
     4141 atgacagagt tagaagatcg cttcaggaag ctatttggca cgacttctac aacgggagac
     4201 agcacagtag attctgaaga tgaacctcct aaaaaagaaa aaagggtgga ctgggatgag
     4261 tattggaacc ctgaaatcga tagcttccag tgctttgtga aacttcgagg agtctctttg
     4321 ttgaggactt ttgagttctc ccttgaggct cccacagata caataaatat ttgagattga
     4381 accctgtcga gtatctgtgt aatctttttt acctgtgagg tctcggaatc cgggccgaga
     4441 acttcgcagc gagctcattg taccgcgaac ttgtttattg cagcttataa tggttacaaa
     4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4561 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     4621 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4681 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4741 agtgaggagg cttttttgga ggcctagact tttgcagaga ccaaattcgt aatcatgtca
     4801 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4861 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4921 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4981 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5041 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5101 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5161 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5221 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5281 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5341 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5401 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5461 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5521 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5581 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5641 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5701 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5761 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5821 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5881 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5941 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6001 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6061 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6121 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6181 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6241 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6301 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6361 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6421 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6481 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6541 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6601 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6661 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6721 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6781 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6841 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6901 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6961 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     7021 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     7081 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     7141 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     7201 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     7261 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     7321 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     7381 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     7441 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     7501 aggcgtgtac ggtgggaggt ctatataagc agagct
//
