LOCUS       sequence-463824-        4311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector for SNAP-p53DD and HRAS(G12V).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4311)
  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 4311)
  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: pENTR-SNAP-p53DD-IRES-HRAS(G12V)
FEATURES             Location/Qualifiers
     source          1..4311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    10..109
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      176..185
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             182..727
                     /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(1208..1225)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1435..1454
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1609..1641,1642..1644,1645..2178)
                     /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"
     protein_bind    complement(2192..2291)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2403..2422)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2414..3223
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2481..2500)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3313..3901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3802..3821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4066..4152
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4244..4271
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     4279..4302
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt
       61 gataagcaat gcttttttat aatgccaact ttgtacaaaa aagcaggctc caccatggga
      121 accaattcag tcgactggat ccggtactac tgaattcaag cttggatccc aatgtgccac
      181 catggacaaa gactgcgaaa tgaagcgcac caccctggat agccctctgg gcaagctgga
      241 actgtctggg tgcgaacagg gcctgcaccg tatcatcttc ctgggcaaag gaacatctgc
      301 cgccgacgcc gtggaagtgc ctgccccagc cgccgtgctg ggcggaccag agccactgat
      361 gcaggccacc gcctggctca acgcctactt tcaccagcct gaggccatcg aggagttccc
      421 tgtgccagcc ctgcaccacc cagtgttcca gcaggagagc tttacccgcc aggtgctgtg
      481 gaaactgctg aaagtggtga agttcggaga ggtcatcagc tacagccacc tggccgccct
      541 ggccggcaat cccgccgcca ccgccgccgt gaaaaccgcc ctgagcggaa atcccgtgcc
      601 cattctgatc ccctgccacc gggtggtgca gggcgacctg gacgtggggg gctacgaggg
      661 cgggctcgcc gtgaaagagt ggctgctggc ccacgagggc cacagactgg gcaagcctgg
      721 gctgggtcct gcaggcggat ccatgactgc catggaggag tcacagtcgg atatcagcct
      781 caagagagcg ctgcccacct gcacaagcgc ctctcccccg caaaagaaaa aaccacttga
      841 tggagagtat ttcaccctca agatccgcgg gcgtaaacgc ttcgagatgt tccgggagct
      901 gaatgaggcc ttagagttaa aggatgccca tgctacagag gagtctggag acagcagggc
      961 tcactccagc tacctgaaga ccaagaaggg ccagtctact tcccgccata aaaaaacaat
     1021 ggtcaagaaa gtggggcctg actcagactg acaacaccat ggagcgttac tggccgaagc
     1081 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     1141 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     1201 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     1261 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     1321 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     1381 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     1441 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     1501 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     1561 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacaggaat gaccgaatac
     1621 aagctggtgg tggtgggcgc cgtcggtgtg ggcaagagtg cgctgaccat ccagctgatc
     1681 cagaaccatt ttgtggacga atacgacccc actatagagg attcctaccg gaagcaggtg
     1741 gtcattgatg gggagacgtg cctgttggac atcctggata ccgccggcca ggaggagtac
     1801 agcgccatgc gggaccagta catgcgcacc ggggagggct tcctgtgtgt gtttgccatc
     1861 aacaacacca agtcttttga ggacatccac cagtacaggg agcagatcaa acgggtgaag
     1921 gactcggatg acgtgcccat ggtgctggtg gggaacaagt gtgacctggc tgcacgcact
     1981 gtggaatctc ggcaggctca ggacctcgcc cgaagctacg gcatccccta catcgaaacc
     2041 tcggccaaga cccggcaggg agtggaggat gccttctaca cgttggtgcg tgagatccgg
     2101 cagcacaagc tgcggaagct gaaccctcct gatgagagtg gccccggctg catgagctgc
     2161 aagtgtgtgc tctcctaacg ctgatatcta gacccagctt tcttgtacaa agttggcatt
     2221 ataagaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     2281 atcattattt gccatccagc tgcagctctg gcccgtgtct caaaatctct gatgttacat
     2341 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     2401 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     2461 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     2521 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     2581 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     2641 tatgcctctt ccgaccatca agcattttat ccgtactcct ggtgatgcat ggttactcac
     2701 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     2761 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     2821 ttgtcctttt aacagcgatc gcgtatttcg tctagctcag gcgcaatcac gaatgaataa
     2881 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     2941 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     3001 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     3061 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     3121 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     3181 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     3241 gttgtaacat tattcagatt gggccccgtt ccactgagcg tcagaccccg tagaaaagat
     3301 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3361 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3421 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3481 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3541 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3601 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3661 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3721 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3781 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3841 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3901 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3961 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcta gcatggatct
     4021 cggggacgtc taactactaa gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     4081 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4141 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     4201 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     4261 gatggccttt ttgcgtttct acaaactctt cctgttagtt agttacttaa g
//
