The composite sequence encompassing the coding sequences of
RAGE signal peptide, the inserted T7 epitope tag, and sRAGE with a
stop codon was optimized using the GeneOptimizer expert software
(Life technologies-Invitrogen Inc.). The optimization was performed
according to the general rationale of gene design [31] to
remove putative RNA secondary structures that may potentially
obstruct ribosomal translation, to eliminate cryptic splice sites,
direct repeats, and RNA destabilizing sequence elements. The program
also optimizes the GC content and distribution, and adjusts
the codon preference to Chinese hamster (Ceanothus griseus). In
addition, a Kozak consensus sequence was integrated into sRAGE
cDNA to facilitate translational initiation in mammalian cells [32].