LOCUS       sequence-465057-        6870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of SNAP-p53DD and HRAS(G12V).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6870)
  AUTHORS   Wang NB, Adewumi HO, Lende-Dorn BA, Beitz AM, O'Shea TM, Galloway KE
  TITLE     Compact transcription factor cassettes generate functional, 
            engraftable motor neurons by direct conversion.
  JOURNAL   Cell Syst. 2025 Mar 10:101206. doi: 10.1016/j.cels.2025.101206.
  PUBMED    40086435
REFERENCE   2  (bases 1 to 6870)
  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"; journalName: "Cell Syst. 
            2025 Mar 10:101206. doi: 10.1016/j.cels.2025.101206."
COMMENT     Sequence Label: pAAV-CAG-SNAP-p53DD-IRES-HRAS(G12V)-bGH
FEATURES             Location/Qualifiers
     source          1..6870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   64..193
                     /label=AAV2 ITR
     protein_bind    206..230
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        335..638
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        640..917
                     /label=chicken beta-actin promoter
     intron          918..1934
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1858..1880
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1942..1961
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1994..2539
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(3020..3037)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3247..3266
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3421..3453,3454..3456,3457..3990)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase H-Ras"
                     /label=H-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    4002..4226
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4240..4264)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   4291..4420
                     /label=AAV2 ITR
     rep_origin      complement(4657..5245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4737..4756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5416..6207,6208..6276))
                     /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     6039..6058
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6277..6381)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6407..6862)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6544..6565)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6756..6775
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
       61 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
      121 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      181 actaggggtt ccttgtagtt aatgaacaag tttgtacaaa aaagcaggct ccaccatggg
      241 aaccaattca gtcgactgga tccggtacta ctactagtta ttaatagtaa tcaattacgg
      301 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      361 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      421 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      481 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      541 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      601 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
      661 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
      721 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
      781 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      841 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
      901 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
      961 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1021 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1081 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1141 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1201 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     1261 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     1321 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     1381 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     1441 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     1501 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     1561 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     1621 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     1681 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     1741 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     1801 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     1861 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     1921 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     1981 aaaatgtgcc accatggaca aagactgcga aatgaagcgc accaccctgg atagccctct
     2041 gggcaagctg gaactgtctg ggtgcgaaca gggcctgcac cgtatcatct tcctgggcaa
     2101 aggaacatct gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc
     2161 agagccactg atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat
     2221 cgaggagttc cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg
     2281 ccaggtgctg tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctacagcca
     2341 cctggccgcc ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg
     2401 aaatcccgtg cccattctga tcccctgcca ccgggtggtg cagggcgacc tggacgtggg
     2461 gggctacgag ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact
     2521 gggcaagcct gggctgggtc ctgcaggcgg atccatgact gccatggagg agtcacagtc
     2581 ggatatcagc ctcaagagag cgctgcccac ctgcacaagc gcctctcccc cgcaaaagaa
     2641 aaaaccactt gatggagagt atttcaccct caagatccgc gggcgtaaac gcttcgagat
     2701 gttccgggag ctgaatgagg ccttagagtt aaaggatgcc catgctacag aggagtctgg
     2761 agacagcagg gctcactcca gctacctgaa gaccaagaag ggccagtcta cttcccgcca
     2821 taaaaaaaca atggtcaaga aagtggggcc tgactcagac tgacaacacc atggagcgtt
     2881 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     2941 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     3001 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     3061 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     3121 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     3181 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     3241 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     3301 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     3361 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacagga
     3421 atgaccgaat acaagctggt ggtggtgggc gccgtcggtg tgggcaagag tgcgctgacc
     3481 atccagctga tccagaacca ttttgtggac gaatacgacc ccactataga ggattcctac
     3541 cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga taccgccggc
     3601 caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg cttcctgtgt
     3661 gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag ggagcagatc
     3721 aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa gtgtgacctg
     3781 gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta cggcatcccc
     3841 tacatcgaaa cctcggccaa gacccggcag ggagtggagg atgccttcta cacgttggtg
     3901 cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc ctgatgagag tggccccggc
     3961 tgcatgagct gcaagtgtgt gctctcctaa caacgttgca cctgtgcctt ctagttgcca
     4021 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4081 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4141 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4201 tgctggggat gcggtgggct ctatggcgct gatatctaga cccagctttc ttgtacaaag
     4261 tggtgcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc
     4321 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4381 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca
     4441 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4501 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4561 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4621 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4681 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4741 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4801 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4861 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4921 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4981 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5041 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5101 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5161 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5221 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5281 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5341 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5401 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5461 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5521 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5581 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5641 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5701 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5761 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5821 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5881 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5941 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6001 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6061 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6121 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6181 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6241 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6301 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6361 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     6421 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6481 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6541 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6601 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6661 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6721 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6781 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6841 aatgcgccgc tacagggcgc gtcccattcg
//
