Cloning of each F8 domain. (A) A1, A2, B, A3, C1, and C2 represent the domains of human FVIII, and the bars indicate the cloning region of each domain. The numbers under each bar indicate the corresponding sequence of amino acids in each domain. (B) pET-28a(+) vectors containing each recombinant F8 domain were digested with BamHI and XhoI. Each domain was subcloned into pGEX4T-2, a GST expression vector. The plasmids were purified, and the products were subjected to BamHI and XhoI digestion. Lane 1, pGEX only; lane 2, pGEX-A1; lane 3, pGEX-A2; lane 4, pGEX-A3; and lane 5, pGEX-C. Arrowheads indicate the products of each domain. M1: 1 kb DNA marker, M2: 100 bp DNA marker.