This PCR item was cloned into pCRBlunt (Invitrogen Life Technologies) according to the manufacturer’s protocols for ligation and E. coli transformation, adopted by Sanger DNA sequencing to verify that no mutations experienced been introduced

pCRBlunt by restriction digestion with HindIII-ClaI and ClaIXbaI, respectively, followed by gel-purification of every insert (DNA Gel Extraction Kit, Zymo Analysis). Plasmid pBT2 was digested with HindIII-XbaI, and a ``triple''…