The amplicons generated by PCR were ligated into pTZ57R/T cloning vector using an InsTAclone™ PCR cloning kit (Fermentas Life Sciences) according to the manufacturer’s instructions, transformed into competent cells by the heat shock method. Recombinant plasmid DNA was isolated from overnight grown liquid cultures of selected clones using the Plasmid Mini Prep Kit (Sigma–Aldrich) or PCR products were directly sequenced