(HDV) ribozyme self-cleavage site, and bovine growth
hormone termination and poly(A) sequence (BGH)
(Fig. 3b). It is important that the synthetic construct
generates mRNA bearing 39 and 59 UTRs identical to
the natural PEDV RNA. Therefore, the plasmid was
checked by transfection into human embryonic kidney
(HEK) 293T cells and the mRNA of GFP was subsequently
extracted and subjected to 39 and 59 RACE analysis to
verify the sequences of the 39 and 59 UTRs transcribed
under the synthesized CMV promoter (data not shown).
Once the plasmid was verified, the GFP gene was then
exchanged with the full-length PEDV fragment generated
from the sequential cloning of fragments A–H according
to the strategy depicted in Fig. 3(c), resulting in the generation
of the full-length infectious cDNA clone, pSMARTBAC-PEDVAVCT12.