Briefly, pMH54
contains a T7 RNA polymerase promoter followed by a 9,139-nucleotide sequence
(11); pMH54 encodes the 5 end of the MHV genome fused in frame to
codon 28 of the hemagglutinin esterase pseudogene, followed by the spike and
the rest of the 3 end of the MHV-A59 genome and finally a poly(A) tail
In the
final pMH54-gp33/EGFPconstruct, the ATG for ORF4a is 50 nucleotides downstream
of the gene 3/gene4 intergenic sequence. This is followed by the nine
codons of ORF4a, the nine codons of gp33, seven intervening codons followed by
the ATG for the EGFP gene, and the rest of the EGFP gene, such that ORF4a,
gp33, and the EGFP protein are all in frame