LOCUS       sequence-472890-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping Beauty transposon plasmid for hydrodynamic tail vein 
            injection (HDTVi) into mice. Pgk 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 6508)
  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 6508)
  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/PGK-mVenus-P2A-NRASG12V
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        472..971
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(507..530)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     904..923
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(983..985,986..988,989..1699)
                     /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="MVSKGEELFTGGGPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1028..1049)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1289..1308)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1636..1657
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1709..1765
                     /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    2391..2964
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2558..2575)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2785..2804
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(3214..3231)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3220..3444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3891..3907)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3891..3907)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3904..3926)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3915..3931
                     /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(3939..3945,3946..3963,3964..3969))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3984..4005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4122..4139)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4293..4881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4373..4392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5052..5843,5844..5912))
                     /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     5675..5694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5913..6017)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6085..6103
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6141..6163)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6263..6282
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6476..6498
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6490..6507
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6491..6507
                     /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 tcgattctac cgggtagggg
      481 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
      541 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
      601 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
      661 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
      721 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
      781 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
      841 ggggcgggct caggggcggg ctcaggggcg gggcgggggc ccgaagggcc tccgggggcc
      901 ccgcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
      961 ccggcctttc gaccgcggcc gcatggtgag caagggcgag gagctgttca ccgggggggg
     1021 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1081 gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa
     1141 gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc agtgcttcgc
     1201 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1261 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1321 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1381 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1441 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     1501 ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1561 cgtgctgctg cccgacaacc actacctgag ctaccagtcc aagctgagca aagaccccaa
     1621 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1681 catggacgag ctgtacaaag gaagcggagc tactaacttc agcctgctga agcaggctgg
     1741 agacgtggag gagaaccctg gacctatgac tgagtacaaa ctggtggtgg ttggagcagt
     1801 tggtgttggg aaaagcgcac tgacaatcca gctaatccag aaccactttg tagatgaata
     1861 tgatcccacc atagaggatt cttacagaaa acaagtggtt atagatggtg aaacctgttt
     1921 gttggacata ctggatacag ctggacaaga agagtacagt gccatgagag accaatacat
     1981 gaggacaggc gaaggcttcc tctgtgtatt tgccatcaat aatagcaagt catttgcgga
     2041 tattaacctc tacagggaac agattaagcg agtaaaagac tcggatgatg tacctatggt
     2101 gctagtggga aacaagtgtg atttgccaac aaggacagtt gatacaaaac aagcccacga
     2161 actggccaag agttacggga ttccattcat tgaaacctca gccaagacca gacagggtgt
     2221 tgaagatgct ttttacacac tggtaagaga aatacgccag taccgaatga aaaaactcaa
     2281 cagcagtgat gatgggactc agggttgtat gggattgcca tgtgtggtga tgtaaaccgg
     2341 taggcctcgt acgcttaatt aacggatccg gaattccgcc cccccccccc cccctctccc
     2401 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     2461 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     2521 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     2581 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     2641 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     2701 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     2761 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     2821 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     2881 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     2941 ttcctttgaa aaacacgatg ataagcttga tcacgcgtct cgagatatca gtggtccagg
     3001 ctctagtttt gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa
     3061 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     3121 ttggcaagct agcattcccg ggaaaactca acagcagtga tgatgggact cagggttgta
     3181 tgggattgcc atgtgtggtg atgtaaggat cagcctcgac tgtgccttct agttgccagc
     3241 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3301 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3361 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3421 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagccat cacaaagctc
     3481 tgacctcaat cctatagaaa ggaggaatga gccaaaattc acccaactta ttgtgggaag
     3541 cttgtggaag gctactcgaa atgtttgacc caagttaaac aatttaaagg caatgctacc
     3601 aaatactaat tgagtgtatg ttaacttctg acccactggg aatgtgatga aagaaataaa
     3661 agctgaaatg aatcattctc tctactatta ttctgatatt tcacattctt aaaataaagt
     3721 ggtgatccta actgacctta agacagggaa tctttactcg gattaaatgt caggaattgt
     3781 gaaaaagtga gtttaaatgt atttggctaa ggtgtatgta aacttccgac ttcaactgta
     3841 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     3901 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3961 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4081 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4141 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4201 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4261 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4321 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4381 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4441 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4501 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4561 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4621 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4681 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4741 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4801 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4861 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4921 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4981 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5041 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5101 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5161 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5221 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5281 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5341 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5401 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5461 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5521 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5581 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5641 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5701 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5761 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5821 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5881 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5941 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6001 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     6061 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     6121 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     6181 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     6241 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     6301 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
     6361 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     6421 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     6481 tcacgacgtt gtaaaacgac ggccagtg
//
