Figure 3

A) A ready-made library of normalized, quality-controlled monomers provides the building blocks for TALE assembly. B) According to the custom TALE design, monomers are assembled into 2–3 multimers in a restriction and ligation-based procedure, using a thermocycler. In the example shown here, multimer 1 and 2 are hexamers while multimer 3 is a tetramer. C) E. coli transformation with the final assembly product in a vector of choice typically results in tens to hundreds of colonies, while the negative control should have significantly lower to no colonies D) Correct assembly of multimers into the vector can be assessed by colony PCR, and further confirmed by sequencing (E). F) As the TALE binding specificity is based on 4 types of RVDs, color-coding the RVD-encoding nucleotides can quickly reveal the correct order of tandem repeats. G) For functional validation, a custom TALE domain was assembled into the EF1-TALE-TF vector. In addition, a dual reporter construct was generated carrying 3 copies of the TALE binding sequence. H) Co-transfection of the custom TALE-TF and the dual reporter construct into HEK293 cells showed strong induction of luciferase activity, confirming the TALE-TF functionality.