LOCUS       sequence-472895-        7718 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 mutant NRAS (NRASG12V)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7718)
  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 7718)
  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-mVenus-P2A-NRASG12V
FEATURES             Location/Qualifiers
     source          1..7718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(462..478)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(463..479)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     enhancer        478..857
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1137..2153
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2077..2099
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2161..2180
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2216..2218,2219..2221,2222..2932)
                     /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(2261..2282)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2522..2541)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2869..2890
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2942..2998
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     misc_feature    3608..4174
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3768..3785)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3995..4014
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4424..4441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4430..4654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5101..5117)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5101..5117)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5114..5136)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5125..5141
                     /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(5149..5155,5156..5173,5174..5179))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5194..5215
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5332..5349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5503..6091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5583..5602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6262..7053,7054..7122))
                     /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     6885..6904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7123..7227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7295..7313
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7351..7373)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7473..7492
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7686..7708
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7700..7717
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7701..7717
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aattcgagct cggtacccta cagttgaagt cggaagttta catacactta agttggagtc
       61 attaaaactc gtttttcaac tactccacaa atttcttgtt aacaaacaat agttttggca
      121 agtcagttag gacatctact ttgtgcatga cacaagtcat ttttccaaca attgtttaca
      181 gacagattat ttcacttata attcactgta tcacaattcc agtgggtcag aagtttacat
      241 acactaagtt gactgtgcct ttaaacagct tggaaaattc cagaaaatga tgtcatggct
      301 ttagaagctt ctgatagact aattgacatc atttgagtca attggaggtg tacctgtgga
      361 tgtatttcaa ggcgatgtac gggccagata tacgcgttga cattgattat tgactagtaa
      421 cggccgccag tgtgctggaa ttctgcagat atcaagctta tcgataccgt cgacctcgac
      481 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      541 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      601 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      781 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      841 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      901 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      961 gggcgggggg gggggggggc gcgcgccagg gggggggggg gggggggggg gggggggggg
     1021 gggggggggg gggggggggg gggggcgcca gggggggggg ggcgcccccg agggttcctt
     1081 tttttggggg gggggggggg ggggggccct ataaaaagcg aagcgcgcgg cgggcgggag
     1141 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     1201 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     1261 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
     1321 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt
     1381 gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg
     1441 ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg
     1501 gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1561 tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc
     1621 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg
     1681 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
     1741 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct
     1801 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1861 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
     1921 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
     1981 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     2041 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     2101 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     2161 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcgcg gccgcatggt
     2221 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2281 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2341 gctgaccctg aagctgatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2401 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     2461 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2521 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2581 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2641 ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
     2701 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
     2761 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2821 gagctaccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2881 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca aaggaagcgg
     2941 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat
     3001 gactgagtac aaactggtgg tggttggagc agttggtgtt gggaaaagcg cactgacaat
     3061 ccagctaatc cagaaccact ttgtagatga atatgatccc accatagagg attcttacag
     3121 aaaacaagtg gttatagatg gtgaaacctg tttgttggac atactggata cagctggaca
     3181 agaagagtac agtgccatga gagaccaata catgaggaca ggcgaaggct tcctctgtgt
     3241 atttgccatc aataatagca agtcatttgc ggatattaac ctctacaggg aacagattaa
     3301 gcgagtaaaa gactcggatg atgtacctat ggtgctagtg ggaaacaagt gtgatttgcc
     3361 aacaaggaca gttgatacaa aacaagccca cgaactggcc aagagttacg ggattccatt
     3421 cattgaaacc tcagccaaga ccagacaggg tgttgaagat gctttttaca cactggtaag
     3481 agaaatacgc cagtaccgaa tgaaaaaact caacagcagt gatgatggga ctcagggttg
     3541 tatgggattg ccatgtgtgg tgatgtaaac cggtaggcct cgtacgctta attaacggat
     3601 ccggaattcc gccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     3661 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     3721 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     3781 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     3841 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     3901 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     3961 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     4021 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     4081 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     4141 ggacgtggtt ttcctttgaa aaacacgatg ataagcttga tcacgcgtct cgagatatca
     4201 gtggtccagg ctctagtttt gactcaacaa tatcaccagc tgaagcctat agagtacgag
     4261 ccatagataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     4321 acctgtaggt ttggcaagct agcattcccg ggaaaactca acagcagtga tgatgggact
     4381 cagggttgta tgggattgcc atgtgtggtg atgtaaggat cagcctcgac tgtgccttct
     4441 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4501 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4561 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4621 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagccat
     4681 cacaaagctc tgacctcaat cctatagaaa ggaggaatga gccaaaattc acccaactta
     4741 ttgtgggaag cttgtggaag gctactcgaa atgtttgacc caagttaaac aatttaaagg
     4801 caatgctacc aaatactaat tgagtgtatg ttaacttctg acccactggg aatgtgatga
     4861 aagaaataaa agctgaaatg aatcattctc tctactatta ttctgatatt tcacattctt
     4921 aaaataaagt ggtgatccta actgacctta agacagggaa tctttactcg gattaaatgt
     4981 caggaattgt gaaaaagtga gtttaaatgt atttggctaa ggtgtatgta aacttccgac
     5041 ttcaactgta ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg
     5101 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5161 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5221 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5281 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5341 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5401 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5461 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5521 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5581 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5641 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5701 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5761 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5821 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5881 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5941 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6001 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6061 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6121 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6181 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6241 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6301 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6361 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6421 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6481 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6541 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6601 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6661 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6721 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6781 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6841 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6901 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6961 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7021 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7081 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7141 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7201 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7261 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7321 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7381 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7441 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7501 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     7561 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     7621 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     7681 gttttcccag tcacgacgtt gtaaaacgac ggccagtg
//
