Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan 
intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, 
X≠P). These enzymes are typically heteromeric complexes composed of several 
membrane-associated subunits, in which STT3 is highly conserved as a catalytic 
core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their 
genome, resulting in the formation of at least two distinct OST isoforms 
consisting of shared subunits and complex specific subunits. The STT3A isoform 
of OST primarily glycosylates substrate polypeptides cotranslationally, whereas 
the STT3B isoform is involved in cotranslational and post-translocational 
glycosylation of sequons that are skipped by the STT3A isoform. Here, we 
describe mutant constructs of monomeric enhanced green fluorescent protein 
(mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The 
endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an 
N-glycosylation sequon at their C-terminus and exhibited increased fluorescence 
in response to N-glycosylation. Isoform-specific glycosylation of the constructs 
was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant 
constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 
sequon was the best substrate for the STT3B isoform in terms of glycosylation 
efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP 
is useful for monitoring STT3B-dependent post-translocational N-glycosylation in 
cells of interest, such as those from putative patients with a congenital 
disorder of glycosylation.