LOCUS       sequence-472893-        7618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping Beauty transposon plasmid for hydrodynamic tail vein 
            injection (HDTVi) into mice. Caggs promotor driven expression of 
            mVenus and a double mutant NRAS (NRASG12VD38A)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7618)
  AUTHORS   Chan ASL, Zhu H, Narita M, Cassidy LD, Young ARJ, Bermejo-Rodriguez 
            C, Janowska AT, Chen HC, Gough S, Oshimori N, Zender L, Aitken SJ, 
            Hoare M, Narita M
  TITLE     Titration of RAS alters senescent state and influences tumour 
            initiation.
  JOURNAL   Nature. 2024 Sep;633(8030):678-685. doi: 10.1038/s41586-024-07797-z.
            Epub 2024 Aug 7.
  PUBMED    39112713
REFERENCE   2  (bases 1 to 7618)
  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/s41586-024-07797-z"; journalName: "Nature"; date: 
            "2024-09"; volume: "633"; issue: "8030"; pages: "678-685"
COMMENT     Sequence Label: pT/CAGGS-NRASG12V/D38A-IRES-mVenus
FEATURES             Location/Qualifiers
     source          1..7618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(229..245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(229..245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(242..264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    253..269
                     /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(277..283,284..301,302..307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    322..343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(460..477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(631..1219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(711..730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1390..2181,2182..2250))
                     /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     2013..2032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2251..2355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2423..2441
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2479..2501)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2601..2620
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2814..2836
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2828..2845
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2829..2845
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3308..3324)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3309..3325)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     enhancer        3324..3703
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          3983..4999
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4923..4945
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5007..5026
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5090..5107
                     /codon_start=1
                     /product="epitope tag from middle T antigen of mouse 
                     polyoma virus"
                     /label=Glu-Glu tag
                     /translation="EYMPME"
     misc_feature    5691..6258
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    5708..6260
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5852..5869)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6079..6098
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(6272..6274,6275..6277,6278..6991)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6317..6338)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6578..6597)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6925..6946
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7170..7187)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7176..7400
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ctgaaatgaa tcattctctc tactattatt ctgatatttc acattcttaa aataaagtgg
       61 tgatcctaac tgaccttaag acagggaatc tttactcgga ttaaatgtca ggaattgtga
      121 aaaagtgagt ttaaatgtat ttggctaagg tgtatgtaaa cttccgactt caactgtagg
      181 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt
      241 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
      301 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
      361 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      421 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
      481 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      541 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      601 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      661 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      721 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      781 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      841 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      901 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      961 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1021 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1081 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1141 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1201 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1261 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1321 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1381 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1441 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1501 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1561 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1621 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1681 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1741 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1801 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1861 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1921 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1981 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2041 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2101 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2161 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2221 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2281 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2341 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     2401 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     2461 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2521 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2581 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     2641 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     2701 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2761 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     2821 acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accctacagt tgaagtcgga
     2881 agtttacata cacttaagtt ggagtcatta aaactcgttt ttcaactact ccacaaattt
     2941 cttgttaaca aacaatagtt ttggcaagtc agttaggaca tctactttgt gcatgacaca
     3001 agtcattttt ccaacaattg tttacagaca gattatttca cttataattc actgtatcac
     3061 aattccagtg ggtcagaagt ttacatacac taagttgact gtgcctttaa acagcttgga
     3121 aaattccaga aaatgatgtc atggctttag aagcttctga tagactaatt gacatcattt
     3181 gagtcaattg gaggtgtacc tgtggatgta tttcaaggcg atgtacgggc cagatatacg
     3241 cgttgacatt gattattgac tagtaacggc cgccagtgtg ctggaattct gcagatatca
     3301 agcttatcga taccgtcgac ctcgacattg attattgact agttattaat agtaatcaat
     3361 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     3421 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     3481 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     3541 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     3601 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     3661 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     3721 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     3781 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg ccccgggggg
     3841 gggggggggg gggggggggg gggggggggc gagggggaga ggtgcggcgg cagccaatca
     3901 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     3961 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc
     4021 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
     4081 cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt
     4141 ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg
     4201 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc
     4261 gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt
     4321 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa
     4381 caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt
     4441 cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg
     4501 tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca
     4561 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg
     4621 cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt
     4681 atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa
     4741 atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg
     4801 caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc
     4861 tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc
     4921 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc
     4981 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt
     5041 tggcaaagaa ttcctcgata ccgggggcgc gtccaccatg gaggaagagg aatatatgcc
     5101 tatggaaatg actgagtaca aactggtggt ggttggagca gttggtgttg ggaaaagcgc
     5161 actgacaatc cagctaatcc agaaccactt tgtagatgaa tatgatccca ccatagaggc
     5221 ttcttacaga aaacaagtgg ttatagatgg tgaaacctgt ttgttggaca tactggatac
     5281 agctggacaa gaagagtaca gtgccatgag agaccaatac atgaggacag gcgaaggctt
     5341 cctctgtgta tttgccatca ataatagcaa gtcatttgcg gatattaacc tctacaggga
     5401 acagattaag cgagtaaaag actcggatga tgtacctatg gtgctagtgg gaaacaagtg
     5461 tgatttgcca acaaggacag ttgatacaaa acaagcccac gaactggcca agagttacgg
     5521 gattccattc attgaaacct cagccaagac cagacagggt gttgaagatg ctttttacac
     5581 actggtaaga gaaatacgcc agtaccgaat gaaaaaactc aacagcagtg atgatgggac
     5641 tcagggttgt atgggattgc catgtgtggt gatgtaagcg gccaacgaat tccgcccccc
     5701 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     5761 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     5821 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     5881 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     5941 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     6001 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     6061 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     6121 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     6181 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     6241 tgaaaaacac gatgataata tggccacaac catggtgagc aagggcgagg agctgttcac
     6301 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     6361 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagc tgatctgcac
     6421 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgggct acggcctgca
     6481 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     6541 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     6601 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     6661 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     6721 cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca
     6781 caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     6841 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtcca agctgagcaa
     6901 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     6961 cactctcggc atggacgagc tgtacaagta agtcgacctg cagccaagct tatcgataaa
     7021 ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt
     7081 tggcaagcta gccccgggaa aactcaacag cagtgatgat gggactcagg gttgtatggg
     7141 attgccatgt gtggtgatgt aaggatcagc ctcgactgtg ccttctagtt gccagccatc
     7201 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     7261 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     7321 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     7381 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agccatcaca aagctctgac
     7441 ctcaatccta tagaaaggag gaatgagcca aaattcaccc aacttattgt gggaagcttg
     7501 tggaaggcta ctcgaaatgt ttgacccaag ttaaacaatt taaaggcaat gctaccaaat
     7561 actaattgag tgtatgttaa cttctgaccc actgggaatg tgatgaaaga aataaaag
//
