Subcellular targeting of opsins in optogenetics provides new possibilities for investigating the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of potential-dependent potassium channel Kv2.1. We have expressed construct CHR2-Venus-Kv2.1 in the layer 2/3 pyramidal neurons of the murine cerebral cortex by means of in utero electroporation. It was found that, although the majority of neurons expressing CHR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence in the soma, proximal dendrites and axon, there was also significant population of neurons with disruption of the “correct” targeting resulting in the fluorescent protein distributed uniformly throughout the entire cell surface. We have suggested that observed mislocalization was caused by overexpression of the construct. Indeed, a decrease in the plasmid concentration during the in utero electroporation procedure resulted in almost complete absence of neurons with altered targeting. Thus, the possibility of “incorrect” targeting of CHR2 by the potassium channel motif Kv2.1 should be taken into account when using this construct in optogenetic experiments.