Pgem 5zf plasmid

TALENs targeted to the CD40LG 5’ untranslated region were designed with the online TAL Effector Nucleotide Targeter 2.0. program(Cermak et al., 2011 (link); Doyle et al., 2012 (link)) and assembled using the Golden Gate TALEN kit (Addgene; Cambridge, MA) and Golden Gate TALEN assembly protocol.(Cermak et al., 2011 (link)) TALENs were subsequently cloned into a pCAG mammalian expression vector. The T7 promoter was cloned into the vector preceding the TALEN sequence using a gene block containing the T7 promoter with the In-Fusion HD Cloning Kit (ClonTech Laboratories; Mountain View, CA). CRISPR single guide RNAs (gRNA) were designed using the online design tool created by the Zhang Lab. Paired oligonucleotides for gRNAs were synthesized (Integrated DNA Technologies; San Diego, CA), annealed, and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 expression vector (Addgene) as previously described(Hsu et al., 2013 (link)) to express both the sgRNA and the Streptococcus pyogenes Cas9. For use as an in vitro mRNA transcription template, hSpCas9 from the pX330 plasmid was cloned into an in-house-modified pGEM-5Zf(+) plasmid (Promega; Madison, WI) that includes optimized 5’ and 3’ UTR sequences and a modified 3’ UTR that encodes a run of 120 adenine nucleotides followed by an SpeI restriction site for linearization.(Warren et al., 2010 (link))