sentence: The pCS-positive plasmid , provided with the kit , was used as the positive control and a sample without sgRNA treatment was used as the negative control.To construct the donor vector , the 873-bp fragment PTCH1-1 and the 473-bp fragment PTCH1-2 , containing the c.403C > T mutation , were joined using overlap PCR to produce the 1.3-kb 5 ′ homologous arm fragment PTCH1-12 , which was then purified and cloned into an intermediate vector , pL452 ( Biocytogen , Beijing , China ) , containing a loxP-flanked PGK-puromycin resistance ( PuroR ) cassette .