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This simple TALE assembly reaction (STAR) strat- egy should enable construction of the repetitive sequence encoding the TALE DNA binding domain from a small 68-part library in just 8 hours (Fig. 1c). Assembly of TALE 4mers is highly efficient using the STAR method. To test the ...
The RFP expression cassette was released by PacI restriction digestion from pEF-RFP and cloned into the resulting vector via the PacI site, generating donor plasmid pGEM-RFP. For construction of the donor plasmid containing RFP and VP2 expression cassettes, the oligo pairs RFP-VP2-F and RFP-...
Construction of the automated imaging system The heated stage consists of an Inheco CPAC Ultraflat HT microplate heater with a temperature range of 4°C to 110°C (Inheco). Microplates were placed on the CPAC and held in place by a custom 3D-printed chamber which incorporates the heated glas...
(Supplementary Figure S6). This is most likely due to 4mer impurities present in the final GA reactions. While less efficient than the TOPO cloning strategy, an efficiency of 5–20% is still sufficient for construction of many sequence-verified TALENs or TALE-TFs in three days including ...