Spherical Proportional Counters (SPCs) are a novel gaseous detector technology employed by the NEWS-G low-mass dark matter search experiment for their high sensitivity to single electrons from ionization. In this paper, we report on the first characterization of the single electron response of SPCs with unprecedented precision, using a UV-laser calibration system. The experimental approach and analysis methodology are presented along with various direct applications for the upcoming next phase of the experiment at SNOLAB. These include the continuous monitoring of the detector response and electron drift properties during dark matter search runs, as well as the experimental measurement of the trigger threshold efficiency. We measure a mean ionization energy of $mathrm{W}=27.6pm0.2~mathrm{eV}$ in $mathrm{Ne + CH_4}$ $(2%)$ for 2.8 keV X-rays, and demonstrate the feasibility of performing similar precision measurements at sub-keV energies for future gas mixtures to be used for dark matter searches at SNOLAB.
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