A full-length cDNA encoding a Crotalus adamanteus venom metalloproteinase II was used (GenBank accession no. JX457344) as a PCR template to subclone its disintegrin domain. PCR was used to generate double stranded cDNA, with the following disintegrin-specific primers (a forward primer 5′-CGCGAATTCGAGGTGGGAGAAGATTGTGACTG-3′ and a reverse primer 5′-GACTCGAGTTAGCCATAGAGGCCATTTCTGGGA-3′, two restriction enzyme sites (underlined): EcoRI in forward primer and XhoI in reverse primer) as previously described (Suntravat et al, 2013 (link)). PCR amplification consisted of a cycle of 94°C (3min), 40 cycles of 94°C (30sec), 60°C (30sec), and 72°C (1min). A final extension step was performed for 10min, at 72°C. The PCR product was digested with EcoRI and XhoI and gel purified. The PCR product was ligated into EcoRI and XhoI sites of pGEX-4T-1 expression vector (GE Healthcare Lifesciences, Uppsala, Sweden), which was a different vector as previously described in Suntravat et al (2013) (link). The ligated plasmid was transformed into E. coli Top10 competent cells (Invitrogen, CA, USA). Plasmid was extracted using the GenElute plasmid miniprep kit (Sigma-Aldrich, MO, USA). Plasmids containing inserts of the predicted size for Cam-dis were performed by PCR and further confirmed by sequencing for construction of in-frame.