TABLE 2.

Primer pairs used in cloning the individual MHV-A59 amplicons

Primer designation	Nucleotide sequencea	Genome location	Orientation	Purpose
	PmeI
MHV A 5′ 17(+)	5′-NGTTTAAACNNNNNNTAATACGACTCACTATAGATAAGAGTGATTGGCGTCCG-3′	1-21	+	T7 start
	CC
MHV A 3520-mu(+)	5′-TAAAGTGGGTTTCATCACTCGGCAC-3′	3528-3506	−	Overlapping PCR Esp3I mutagenesis
	GG
MHV A 3520-mu(−)	5′-GCCGAGTGATGAAACCCACTTTAAAGTG-3′	3506-3533	+	Overlapping PCR Esp3I mutagenesis
	Esp3IGA
MHV A 4870(−)	5′-CGTCTCATTTGTTAACCAGTCGCCAGTCTGTGGGCAG-3′	4887-4856	−	MHV A/B junction
MHV B 4870(+)	5′-CGTCTCACAAATTTGATAGTGTTGATGGTG-3′	4888-4905	+	MHV A/B junction
MHV B 9620(−)	5′-ACTGAACTCGCCGTTAAGGC-3′	9571-9552	−	MHV B/C junction
	T
MHV C 8933(+)	5′-GGTATAGTCTACATTTGGTC-3′	8932-8952	+	MHV B/C junction
MHV C 11511(−)	5′-CGTCTCTGGTTTATGCTACGCATGGTAAC-3′	11515-11493	−	MHV C/D junction
MHV D 11511(+)	5′-CGTCTCAAACCACGACGTCTTTTCTATTATG-3′	11510-11534	+	MHV C/D junction
MHV D 12960(−)	5′-CGTCTCTTAGGATAGCATACACAATCTTACCC-3′	12966-12941	−	MHV D/E junction
	AT
MHV E 12960(+)	5′-CGTCTCTCCTAAGTGACTGTGATGGTCTCAAG-3′	12961-12986	+	MHV D/E junction
MHV E 15760(−)	5′-CGTCTCTAGACATTAGAGTATAGGCGC-3′	15759-15739	−	MHV E/F junction
MHV F 15760(+)	5′-CGTCTCTGTCTATCGTGCGGACCATGTTG-3′	15754-15776	+	MHV E/F junction
	CC
MHV F 17135-mu(−)	5′-TATTCTGAAAGGTTTCAGGCACACTATAAACAC-3′	17150-17118	−	Overlapping PCR Esp3I mutagenesis
	GG
MHV F 17135-mu(+)	5′-GTGCCTGAAACCTTTCAGAATAATGTGCC-3′	17129-17157	+	Overlapping PCR Esp3I mutagenesis
	AA
MHV F 22740(−)	5′-CGTCTCTCGGACCGACTGTCACCAAATAGAAACC-3′	22744-22717	−	MHV F/G junction
	Esp3ITT
MHV G 22760(+)	5′-(N)6CCCGGGCGTCTCGTCCGACTGTACCTATGTAG-3′		+	MHV F/G junction
MHV G 24031(−)	5′-AACGGTCTCAGTGCTAATGCTTGG-3′	24054-24031	−	MHV G end

^aUnderlined sequences represent the T7 start site, and boldface sequences represent the Esp3I or PmeI restriction sites. All primers were derived from the MHV-A59 sequence (30).