The pcgf1 TALEN target site was selected using the online TAL Effector-Nucleotide Targeter tool (https://tale-nt.cac.cornell.edu/ ; [35 (link)]) in exon 2 with the following parameters: (i) spacer length of 14–17 bp, (ii) repeat array length of 16–18 bp, (iii) each binding site was anchored by a preceding T base in position ‘‘0” as has been shown to be optimal for naturally occurring TAL proteins [36 (link), 37 (link)], (iv) presence of a restriction site (ClaI) within the spacer sequence for screening and genotyping purposes.
Pcgf1-specific TALEN constructs were engineered using the TALEN Golden Gate assembly system described by Cermak et al., [38 (link)]. The TALEN expression backbones, pCS2TAL3DD and pCS2TAL3RR [27 (link)], and the plasmids providing repeat variable diresidues (RVD) [38 (link)] for Golden Gate Cloning were obtained from Addgene.
Pcgf1-specific TALEN constructs were engineered using the TALEN Golden Gate assembly system described by Cermak et al., [38 (link)]. The TALEN expression backbones, pCS2TAL3DD and pCS2TAL3RR [27 (link)], and the plasmids providing repeat variable diresidues (RVD) [38 (link)] for Golden Gate Cloning were obtained from Addgene.
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