The N TAILW518 gene construct, encoding residues 401-525 of the MV N protein with a Tyr ! Trp substitution at position 518 and with a hexahistidine tag fused to its N terminus, was obtained by PCR, using the plasmid pET21a/N as template. Two separate PCR steps were carried out in parallel, yielding amplification products A and B. Product A was obtained using forward primer (5 0 -gatagaaccatgCATCAT CATCATCATCATactactgaggacaagatcagtaga-3 0 ), designed to introduce a hexahistidine tag-encoding sequence (upper case) at the N terminus of N TAIL , while reverse primer (5 0 -aaga tttctgtcattCCAcactatTggggtgtc-3 0 ) was designed to introduce a Trp at position 518 (bold and upper case) and a silent mutation at nucleotide position 1545 (upper case) thus resulting in the introduction of a BstXI site (underlined). Product B was obtained using forward primer (5 0 -gacaccccAatagtgTGG aatgacagaaatctt-3 0 ) designed to introduce a Trp at position 518 (bold and upper case) and a silent mutation at nucleotide position 1545 (upper case) thus resulting in the introduction of a BstXI site (underlined), and reverse primer (5 0 -ccgggcatgc atccggatatagttcctcctt-3 0 ) designed to anneal with nucleotide positions 1755-1775 of pet21a/N (where the A of the ATG codon of the N ORF was set as nucleotide position 1). A further PCR step was carried out using amplification products A plus B as template, to yield the N TAIL W518 amplification product. Forward primer (5 0 -ggggacaagtttgtacaaaaaagcaggct tcgaaggagatagaaccatgCATCATCATCAT-3 0 ) was designed to introduce an AttB1 site (bold), while reverse primer (5 0 -ggggac cacttttgtacaagaaagctgggtcttagtctagaagatttctgtcattCCA-3 0 ) was designed to introduce an AttB2 site (bold) and a Trp at position 518 (upper case). After purification, the PCR product was cloned into the pDest14 vector using the Gateway recombination system. The final construct is referred to as pDest14/ N TAILW518 . Candidate clones bearing the desired mutation were selected on the basis of the ability of their recombinant plasmids to be restricted by BstXI, a unique restriction site introduced by PCR together with the Tyr to Trp substitution.