PCR primers used for amplification of opa genes from N. meningitidis and construction of Δopa plasmids.

Restriction sites within primer sequences are underlined. nmb nomenclature as defined in the published sequence of N. meningitidis strain MC58 (GenBank accession number AE002098), which was used to design primers in this study since the sequence of H44/76 had not been published.*nmb0443 is adjacent to opaA but no unique primer site could be identified within nmb0443 so nmb0444 was used;†nmb1635 is adjacent to opaB but the putative coding sequence is only 222 bp so nmb1634 was used;‡nmb1466 is adjacent to opaD but primers within ppx have been published following successful use;§designed during this study;¶from Hobbs et al.[75];∥from Morelli et al.[76];**from Maiden et al.[77];††EryR = erythromycin resistance cassette, which contains the gene ermC;‡‡DNA uptake sequences within NmnDUS5 and NmnDUS3 are italicised;***TetR = tetracycline resistance cassette, which contains the genes tetA and tetR;∥∥product was 584 bp when amplifying Δopa and 1776 bp when amplifying Δopa::ery.