Modular CRR Coding Sequence Assembly

pAR008 allows assembly of a custom CRR coding sequence into a truncTALE (Tal2h) backbone coding sequence using our previously described Golden Gate kit for custom TAL effector construct assembly (Cermak et al., 2011 (link)). The vector was created in two steps with the NEBuilder HiFi DNA assembly kit (New England Biolabs, Ipswich, MA). First, Golden Gate entry vector pTAL1 (Cermak et al., 2011 (link)) was used as a template in a PCR reaction with primer pairs B1778/B1779 and B1781/B1782. The resulting intermediate vector was used as template in a second round of PCR with primer pair B2149/B2150, and Gibson assembled with coding sequence for the Tal2h N-terminal region amplified from genomic subclone pYH2 using primer pair B2147/B2148. Entry vector pAR009 is a version of pTAL1 that contains a silent mutation to abolish the SphI site in the tal1c 3′ end, modified using Q5 mutagenesis (New England Biolabs) with primer pair B2171/B2172. Entry vector pAR012 is pAR008 further modified with a stop codon just prior to the coding sequence for the candidate NLS of Tal2h (RKRKSHD). It was generated in a Q5 mutagenesis reaction with primer pair B2168/B2169. An SphI site and an AatII site in each of these vectors can be used together to shuttle CRR coding sequences from existing TAL effector clones.