Schematic representation of the modular vectors generated and the assembly of binary destination vectors.

<p><b>(A)</b> The modular vectors were designed for overexpression in plants, consisting of FS (flanking sequences), promoter, TALE, FokI and Ter (terminator). The expression of the TALEN cassettes of the modular vectors is driven by the promoters specified below. The CaMV35S promoter (double-enhancer version) and maize ubiquitin promoter were used for dicot or monocot plant transformation. A PacI endonuclease site was embedded into the FS used to obtain DNA fragments. The typical protein structure of an artificial TALEN composed of a repetitive DNA binding domain flanked by an N-terminal and a C-terminal was followed by the cleavage domain of FokI nuclease. The GoldyTALEN scaffold with N- and C-terminal truncations (N152/C63) fused with enhanced heterodimeric (ELD, KKR mutations) FokI domains is also provided. (<b>B</b>) A modified LIC method was implemented to enable high-throughput cloning of constructs for genome editing. The binary destination vector (pTALEN-DES) and DNA fragments containing TALEN cassettes were obtained using the restrictive endonuclease (RE) PacI and were processed by T4 DNA polymerase to create specific 12 nucleotide single-stranded overhangs. After annealing, a circular plasmid was generated in <i>Escherichia coli</i> cells without the use of T4 DNA ligase. (<b>C</b>) A schematic illustrating the construction of the binary destination vector containing four TALEN cassettes. RE, Restriction endonuclease; LB and RB, left border and right border of T-DNA; NptII, neomycin phosphotransferase (KanR); PacI, AgeI, SpeI, SbfI, recognition sequences of the restriction enzymes PacI, AgeI, SpeI and SbfI, respectively.