Figure 2.

Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet, tetracycline resistance; spec, spectinomycin resistance; amp, ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files (Supplementary Data).