Context‐dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

SUMMARY: Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast‐optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context‐aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post‐transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass‐bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5′atpA, and 3′ rbcL in a sense‐transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play. Significance Statement: In this work, we develop a functional fluorescent reporter for Chlamydomonas chloroplast expression, and found construct context‐effects influencing gene expression through formation of antisense dsRNA. These findings will aid in developing more efficient chloroplast genetic strategies and illuminate aspects of chloroplast gene expression. [ABSTRACT FROM AUTHOR]