U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct. A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.