Chapter 5 —— 140 —— Map and nucleotide sequence of selector AAV donor construct BI17_pAAV-HRS1.A1. Diagram of AAV-HRS1.A1 vector genome containing an AAVS1-targeting HR donor template (1966 bp). The selector donor DNA sequence (964 bp) is designed for installing the ouabain resistance gain-of-function Q118R and N129D SNPs (RD), vertical red dashes, within the exon 4 of ATP1A1 upon gRNA GA1-directed cleavage within intron 4. AAV ITR, adenoassociated virus type-2 inverted terminal repeat; orange bars, homology arms (“Left” and “Right”) consisting of DNA homologous to sequences flanking the AAVS1 target site of gRNA GS1; hPGK, human phosphoglycerate kinase 1 gene promoter; EGFP, enhanced green fluorescence protein reporter; bGH pA, bovine growth hormone gene polyadenylation signal; U6 promoter, human U6 snRNA polymerase III promoter driving the expression of each gRNA. The vector plasmid backbone is not shown. tgtaattcagcatatcgatttgtagtacacatcagatatcttctccgtctttgtctcccacttcttctcaatta ccactcattacttaatggttatgaactcattacttaatggttatgaacagctgttgccttcaaggctcatccat tcttccttcgtttccatttcctctctctaccacccacgttgtagatgctcttacaagtgggatgcccacctgca tgtgctgctgtgagcaggcagctctgctcaggccccggccgccacccactggatggcagagcacagcgattcat gttggcacatccacctgtccagaaatgcttggtggccattcttcaaaatccacaagttggttgaaaaacaatct ttgttttataaagcaggagaaactgatgcatctagaacctttccaaacgtccagttagtgatcaagtgttggtg tgcctgactccatttctgacccttcctgtgtggtctttagaagggaattggatcctagagtccggccgccccct tcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattgga attaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtt tgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgg ctttatatatcttgtggaaaggacgaaacaccgagttctgtaattcagcatagtttcagagctatgctggaaac agcatagcaagttgaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaa ttgatccatatatagggcccgggttataattacctcaggtcgacgtcccatgtgcaggtgctgaattgtagata agtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgct cgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcga gcgagcgcgcag
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