1. AAV viral constructs and Rep expression plasmids.
AAV viral constructs and Rep expression plasmids. (A) pTAV2‐0 consists of the entire AAV‐2 genome, including both inverted terminal repeats (TR). pTAV2‐1 is derived from pTAV2‐0, but has the Rep52/40 translation start site methionine 225 mutated to glycine. Spliced and unspliced gene products expressed from the rep open reading frames (ORFs) of the two virus genomes are also shown. AAV promoters are denoted by p5, p19 and p40. (B) The Rep78, Rep68, Rep52 and Rep40 proteins are individually expressed from a CMV promoter within the pKEX‐XL plasmid (Hörer et al., 1995). The K340H small Rep mutants contain a lysine to histidine mutation at amino acid 116 (corresponding to amino acids 340 of the complete Rep ORF), which mutates the ATP binding site (Chejanovsky and Carter, 1990). M225 is also based on pKEX‐XL and allows expression of both Rep52 and Rep40, as the splice sites have not been altered. A series of helicase mutations (E379Q, E379K, K391I, K391T, K404I, K404T) were introduced into M225 as described in Materials and methods. SD− denotes a mutated splice donor site.
Jason A. King et al. EMBO J. 2001;20:
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