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. 2022 Sep 17;50(17):9663–9674. doi: 10.1093/nar/gkac792

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Engineered mutations in Kod-RSGA. (A) The overall architecture of Kod-RSGA TNA polymerase (gray) bound to a DNA duplex (black) is shown with the positions of the amino acid mutations observed in Kod-RI and Kod-RSGA highlighted as green and blue spheres, respectively. Mutations boxed in yellow and gray occur in the finger and thumb subdomains, respectively. (B) A sequence alignment highlights the secondary structures and positions of the engineered mutations. Both mutations in the finger are part of a conserved sequence known as motif B. (C) A time course demonstrates a faster synthesis rate for the Kod-RSGA. Both enzymes extended a 5′ IR680-labeled DNA primer annealed to a DNA template with ptNDP substrates. A DNA primer (11 nt) and a full-length DNA marker (18 nt) are provided. The reaction contained 0.5 μM IR800-labeled primer–template duplex, 100 μM ptNDPs and 0.5 μM Kod-RI or Kod-RSGA (pre-primed with 1 mM MnCl₂), in 1× ThermoPol^® buffer for the 15-min intervals at 55°C.