Transcriptional Activators of Human Genes with Programmable DNA-Specificity (original) (raw)
Figure 3
Golden TAL Technology for assembly of TAL proteins with programmed repeat composition.
(A) Single TAL repeats were cloned with flanking _Bpi_I sites that generate specific four base pair-overhangs (a–g). Matching sites are indicated by identical letters. A library was constructed with four different repeat types (RVD, repeat variable di-residue: NI, HD, NN, NG) for each repeat position. The repeat types have different DNA-specificities (Spec., only upper DNA-strand is shown). (B) One to six repeats are assembled in specific order into an assembly vector to generate a repeat assembly with flanking _Bsa_I sites. (C) TALs were directly assembled with N-terminal GFP-tag into an expression vector using fragments with matching _Bsa_I-generated overhangs (capital letters). Insertion of one to four repeat assemblies generated TALs with 1 to 24 repeats. The last repeat is only a half repeat as typical for TAL proteins. Please see the Text S1 and Figure S4 for details.