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. 2020 Jul 29;5(31):19702–19714. doi: 10.1021/acsomega.0c02396

Figure 1.

Figure 1

Schematic of the new TALEN protocol. (A) Schematic of monomers in a 96-well PCR plate. There are a total of 60 base-determinant monomers and 2 linker monomers (dsDNA10.5 and dsDNA17.5). A TALEN backbone (TALE-FokI) plasmid was also constructed. (B) Structure of TALENs expression plasmid. (C) Structures of linear monomers and the TALEN backbone plasmid. All monomers can be reproduced by a 96-well PCR amplification using a pair of universal primers. CMV: cytomegalovirus promoter; N and C, nonrepetitive amino and carboxyl terminals of TALEN; BsmBI: the cutting site of type IIs restriction enzyme BsmBI used for inserting the custom TALEN DNA-binding domain; LacZ: LacZ expression cassette for a blue-white screen; NLS: nuclear localization signal; FokI: catalytic domain from the FokI endonuclease. (D) Schematic of DNA cut with TALEN (left and right TALENs), which can produce site-specific double-strand breaks (DSB) that can enhance DNA HDR. (E) Pipeline for constructing custom TALEN with linear monomers and the TALEN backbone plasmid. Time used in each step was given. The whole procedure can be completed in one day. TALENs ready to transfect mammalian cells can be obtained in one day. GG, Golden Gate.