Figure 3

Agarose gel image showing PCR amplicons from different steps of Golden Gate assembly. pFUS_A vectors contain 1st-10th RVDs repeats (Lane 1) and pFUS_B5 vectors harbor 11th-15th RVDs repeats (Lane 2) in Golden Gate assembly round 1. The smeared ladder bands starting at ~300 bp with increase of approximate every 100 bp indicated successful RVD assembly and ligation to pFUS_A/B. Unsuccessful insertion led to incorrect size of amplicons (Lane 3). Lane 4 and 5 represent colony PCRs for pCS2-TALEN-ELD/KKR vectors carrying RVDs in Golden Gate assembly round 2. Correct PCR fragments amplified by primer pair of CTalF/CTalR are about 200 bp after assembly of the RVDs repeats from pFUS_A, pFUS_B5, and final half RVD repeat, into the pCS2-TALEN-ELD/KKR vectors.