Cells were imaged and analyzed for steady‐state protein expression level on the Perkin Elmer Operetta System and Columbus software. Transfected cells fixed and DAPI stained on 24 well plates were imaged at ×20 magnification with 60 images taken per variant, in triplicate, for a total of n = 180 images analyzed per variant. Each image was analyzed for cell number via DAPI staining, transfected cell number via mCherry fluorescence, and G6PC‐expressing cell number via EGFP fluorescence. Output was given as percent of transfected (mCherry positive) cells expressing detectable levels of G6PC (EGFP positive) at a preset threshold determined from background levels in negative controls, and compared to WT using Welch's two‐tailed t tests for statistical significance.

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