TABLE 1.

Oligodeoxynucleotide properties

Oligo designator	Direction	Sequence (5′—3′)	Length (bp)	% A/T	Tm (°C)	NEB Tm (°C)
K103C C	Forward	CAAATCAATATGACGATTTTTTTATTTGTATAAGTACTATGACAGATTTTTGAC	54	84	59.8	69
T559C C	Forward	AACAGGACTTAGCCTTGAAAAACTAATAAGATTTTGCACAATTTCTCTTGGATGGGA	57	65	66	77
K591C C	Forward	TGCTATGGAAGTGCTTTCTTGCAATTCTTGCATAGCCTACAGCGGAAG	48	54	67.9	88
K103C NO	Forward	TAGTACTTATACAAATAAAAAAATCG	26	81	56	58
	Reverse	TGACAGATTTTGACTTTAATAAAG	24	75	56	60
T559C NO	Forward	GAGAAATTGTGCAAAATCTTATTAG	25	72	58	62
	Reverse	TTGGATGGGATTCAAATAAC	20	65	57	62
K591C NO	Forward	TGTAGGCTATGCAGAATTGCAAG	23	57	66	70
	Reverse	GCGGAAGCTAACAGCAAAAG	20	50	65	70

All oligos used in this study were purchased from Integrated DNA Technologies (Skokie, IL, USA) with standard desalting and no further purification. These oligos are denoted by the substitution mutation they encode (K103C/T559C/K591C) within p66 and by their design (C, complementary; NO, nonoverlapping). The information provided by this table includes: the oligo sequence, length in base pairs (bp), the percent A/T content, and the calculated melting temperature (Tm). For the complementary oligos, only the forward sequence and corresponding data are included because the reverse oligos are complementary in sequence and identical in the other parameters. The complementary oligos were selected for their range in A/T content and were designed using the QuikChange Primer Design software (https://www.genomics.agilent.com/primerDesignProgram.jsp). The nonoverlapping primers were used for the NEB Q5 SDM protocol and were designed by the NEBaseChanger program [v1.2.7] (https://www.nebasechanger.neb.com). The NEB Tm column contains the suggested melting temperatures for using a given oligo specifically in the Q5 master mix (https://tmcalculator.neb.com).