Construction of JV 3C mut /polySTOP was as follows: complementary oligonucleotides with three translation termination codons (underlined) in each reading frame in sense and anti-sense orientations were desgined in such a way that upon annealing the duplex would contain blunt termini. The oligonucleotides were termed pSTOP Top (59-CTAGGTAAGTAAACGCGTCTACT-CACTCAC-39) and pSTOP Comp (59-GTGAGTGAGTA-GACGCGTTTACTTCAATAG-39). Each oligo (1 mg) was incubated with T4 polynucleotide kinase and ATP to phosphorylate the 59 termini, pooled and heated to 75uC for 15 min, and left to cool to room temperature to anneal the oligos. Following purification the polySTOP duplex was ligated to Eco47III digested JV 3C mut , and ligated DNA was used to transform E.coli Top10. The duplex contained the unique restriction site MluI (shown in bold) to assist screening of recombinant clones.