. 2022 Dec 13;11(1):e03872-22. doi: 10.1128/spectrum.03872-22

TABLE 2.

Primers for constructing transfer vectors used to rescue recombinant PEDVs containing chimeric S genes

Recombinant virus	Transfer vector	Amplified fragment^a	Template	Primer name	Primer location (positions)^b	Primer sense	Primer sequence (5′–3′)
r-S^par	p-PEDV-S^par-ΔORF3/RLuc	PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped138	−42 to −1	−	TTGTTTACGTTGACCAAATGATTAGAAAAGCCACAAATGGCG
		PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped139	End +1 to end +42	+	GCTAGCCACCATGACTTCGAAAGTTTATGATCCAGAACAAAG
		1b′-S^par-RLuc′	1b′-S^par-RLuc′	Ped140	−42 to −1	+	CGCCATTTGTGGCTTTTCTAATCATTTGGTCAACGTAAACAA
		1b′-S^par-RLuc′	1b′-S^par-RLuc′	Ped141	End +1 to end +42	−	CTTTGTTCTGGATCATAAACTTTCGAAGTCATGGTGGCTAGC

r-S^att(1–529 aa)^par	p-PEDV-S^att(1–529 aa)^par-ΔORF3/RLuc	PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped138	−42 to −1	−	TTGTTTACGTTGACCAAATGATTAGAAAAGCCACAAATGGCG
		PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped139	End +1 to end +42	+	GCTAGCCACCATGACTTCGAAAGTTTATGATCCAGAACAAAG
		1b′-(1–529 aa)^par′	1b′-S^par-RLuc′	Ped140	−42 to −1	+	CGCCATTTGTGGCTTTTCTAATCATTTGGTCAACGTAAACAA
		1b′-(1–529 aa)^par′	1b′-S^par-RLuc′	Ped151	1598 to 1620	−	ACAGAAAGAACTAAACCCATTGA
		(530–1381 aa)^att′-RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped146	1586 to 1610	+	CTGACACTACTATCAATGGGTTTAG
		(530–1381 aa)^att′-RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped141	End +1 to end +42	−	CTTTGTTCTGGATCATAAACTTTCGAAGTCATGGTGGCTAGC

r-S^att(530–936 aa)^par	p-PEDV-S^att(530–936 aa)^par-ΔORF3/RLuc	PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped138	−42 to −1	−	TTGTTTACGTTGACCAAATGATTAGAAAAGCCACAAATGGCG
		PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped139	End +1 to end +42	+	GCTAGCCACCATGACTTCGAAAGTTTATGATCCAGAACAAAG
		1b′-(1–529 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped140	−42 to −1	+	CGCCATTTGTGGCTTTTCTAATCATTTGGTCAACGTAAACAA
		1b′-(1–529 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped151	1598 to 1620	−	ACAGAAAGAACTAAACCCATTGA
		(530–936 aa)^par′	1b′-S^par-RLuc′	Ped146	1586 to 1610	+	CTGACACTACTATCAATGGGTTTAG
		(530–936 aa)^par′	1b′-S^par-RLuc′	Ped147	2786 to 2810	−	ACCATGACACCAGAGTAATACTGCG
		(936–1381 aa)^att′-RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped154	2786 to 2810	+	CGCAGTATTACTCTGGTGTCATGGT
		(936–1381 aa)^att′-RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped141	End +1 to end +42	−	CTTTGTTCTGGATCATAAACTTTCGAAGTCATGGTGGCTAGC

r-S^att(936–1381 aa)^par	p-PEDV-S^att(937–1381 aa)^par-ΔORF3/RLuc	PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped138	−42 to −1	−	TTGTTTACGTTGACCAAATGATTAGAAAAGCCACAAATGGCG
		PEDV′-ΔS^att-ΔORF3/RLuc′	p-PEDV-S^att-ΔORF3/RLuc	Ped139	End +1 to end +42	+	GCTAGCCACCATGACTTCGAAAGTTTATGATCCAGAACAAAG
		1b′-(1–936 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped140	−42 to −1	+	CGCCATTTGTGGCTTTTCTAATCATTTGGTCAACGTAAACAA
		1b′-(1–936 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped147	2786 to 2810	−	ACCATGACACCAGAGTAATACTGCG
		(530–1381 aa)^par′-RLuc′	1b′-S^par-RLuc′	Ped154	2786 to 2810	+	CGCAGTATTACTCTGGTGTCATGGT
		(530–1381 aa)^par′-RLuc′	1b′-S^par-RLuc′	Ped141	End +1 to end +42	−	CTTTGTTCTGGATCATAAACTTTCGAAGTCATGGTGGCTAGC

r-S^par(530–936 aa)^att	p-PEDV-S^par(530–936 aa)^att-ΔORF3/RLuc	PEDV′-ΔS^att-ΔORF3/RLuc′	1b′-S^par-RLuc′	Ped138	−42 to −1	−	TTGTTTACGTTGACCAAATGATTAGAAAAGCCACAAATGGCG
		PEDV′-ΔS^att-ΔORF3/RLuc′	1b′-S^par-RLuc′	Ped139	End +1 to end +42	+	GCTAGCCACCATGACTTCGAAAGTTTATGATCCAGAACAAAG
		1b′-(1–529 aa)^par′	1b′-S^par-RLuc′	Ped140	−42 to −1	+	CGCCATTTGTGGCTTTTCTAATCATTTGGTCAACGTAAACAA
		1b′-(1–529 aa)^par′	1b′-S^par-RLuc′	Ped151	1598 to 1620	−	ACAGAAAGAACTAAACCCATTGA
		(530–936 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped146	1586 to 1610	+	CTGACACTACTATCAATGGGTTTAG
		(530–936 aa)^att′	p-PEDV-S^att-ΔORF3/RLuc	Ped147	2786 to 2810	−	ACCATGACACCAGAGTAATACTGCG
		(936–1381 aa)^par′-RLuc′	1b′-S^par-RLuc′	Ped154	2786 to 2810	+	CGCAGTATTACTCTGGTGTCATGGT
		(936–1381 aa)^par′-RLuc′	1b′-S^par-RLuc′	Ped141	End +1 to end +42	−	CTTTGTTCTGGATCATAAACTTTCGAAGTCATGGTGGCTAGC

“′” indicates that the DNA fragment contains homologous arm sequences (flanking sequences) required for subsequent homologous recombination.

The location of the primers is relative to the S gene sequence of the PEDV DR13^par strain (GenBank accession number DQ862099).