Table 1.

List of primers, plasmids and constructs used in this work.

Constructa	Primer #	nt positionb		Deoxyoligonucletide sequence (5′–3′)c	Purpose
		Start	Finish
pJS-21	1	1	38	TGTAATACGACTCACTATAGGTAAAATACCAACTAATTCTCGTTCGATTCCGGCGAAC	To create sgRNA3a (JS-21) that carries 21 A residues at the 3′ end and an additional (to BamHI at 120 nts) marker restriction site PstI at position 1190 nts (underlined). T7 promoter is in bold.
	2	1232	1166	CGCTGAATTAGGACATAGATCTTTTTTTTTTTTTTTTTTTTTAATAATAACTGCAGACACACAACATAGAATATCC
pJS-14	1	See above		See above	To create sgRNA3a (JS-14) that carries 14 A residues at the 3′ end and an additional (to BamHI at 120 nts) marker site PstI at position 1190 nts (underlined).
	3	1232	1166	CGCTGAATTAGGACATAGATCTTTTTTTTTTTTTTAATAATAACTGCAGACACACAACATAGAATATCC
pJS-22	1	See above		See above	To create sgRNA3a (sg-21) that carries 21 A residues at the 3′ end and an additional (to BamHI at 120 nts and PstI at 1190 nts) marker restriction site: HindIII at position 780 nts (underlined).
	4	824	767	GATCCACAGACTGGTTAGAAATACCTCTAATATAATTTTTTAAGCTTTTCTTATCGAG
sgRNA3a-5′	5	32	73	GGCGAACATTCTATTTTACCAACATCGGTTTTTTCAGTAGTG	To clone the full-length segment of sgRNA3a carrying restriction marker sites as a result of RNA3–sgRNA3a recombination.
sgRNA3a-3′	6	1279	1238	GTCATCTTACCAGTTCCTGAAGTCGACATTATTAATACGCTG
pB3-33del	7	70	100	GTGATACTGTTTTGGATCCCGATGTCTAAC	5′ primer, BamHI site (underlined).
	8	530	377	GTAAAGCCGACAACTGAATTGTGGCCTCCTG (500)—(400)G AGAAAACAAACGATGCGTGGAACG	Reverse primer with flanking sequences securing the 33-aa deletion within the 3a ORF
	9	872	843	CTTCCTGGGCAACCTGATCAACAGATTGTAACGGTC	3′ primer, BclI site (underlined)
	10	890	863	CAACTAACAAATCTTCCTGGGCAACCTG	3′ primer, for down- stream to PflMI site.
sg3a-384	2	See above		See above	To amplify the RNA3 cDNA from positions 858 to 1242 (T7 promoter is in bold) and to use it as RNA transcription template.
	11	1242	858	TGTAATACGACTCACTATAGGTTGATCAGGTTGCCCAGGAAG
5′-580	1	See above		See above	To create 3′-nested deletion fragment 580 bp carrying an additional (to BamHI at 120 nts) AvaI marker sites at position 560 nts (underlined).
	12	602	545	ACATCGATTCCTACCGCTATCACCGCCGATGACTTCCATCGGACAATCATAGCTCGGGGTCAA
5′-380	1	See above		See above	To create 3′-nested deletion fragment 380 bp carrying an additional (to BamHI at 120 nts) SmaI marker site at position 360 nts (underlined).
	13	365	333	GAATCCCCTCCCGGGTAACTCTCCTTTATCATACTTGG
5′-150	1	See above		See above	To create 3′-nested deletion fragment 150 bp carrying BamHI restriction site at 120 nts.
	14	142	123	GCCAACGTCAGACGTAGTTCGTGAGG
3′-860	15	1258	1221	CCTGAAGTCGACATTATTAATACGCTGAATTAGGACATAGATC	To create 5′-nested deletion fragment 860 bp, carrying an additional (to PstI at 1190 nts) SmaI marker site at position 360 nts (underlined). T7 promoter is in bold.
	16	352	373	TGTTAATACGACTCACTATAGGAGTTACCCGGGAGGGGATTCATG
3′-680	15	See above		See above	To create 5′-nested deletion fragment 680 bp, carrying an additional (to PstI at 1190 nts) AvaI marker site (underlined). T7 promoter is in bold.
	17	542	564	TGTTAATACGACTCACTATAGGAGCTTTGACCCCGAGCTATGATTG
3′-440	15	See above		See above	To create 5′-nested deletion fragment 440 bp, carrying an additional (to PstI at 1190 nts) HindIII marker site at position 780 nts (underlined). T7 promoter is bold.
	18	757	798	TGTTAATACGACTCACTATAGGA CTGAGACAACTCGATAAGAAAAGCTTAAAAAATTATATTAG
Neg-RNA3	19	1	35	GTAAAATACCAACTAATTCTCGTTCGATTCCGGCG	To create template for in vitro transcription of (−)-strand RNA3 construct. Two rounds of PCR included (i) amplification of RNA3 fragment without T7 promoter with primers 19 and 20 followed by (ii) creation of (−)-strand RNA3 bearing T7 promoter (bold) with primers 19 and 21.
	20	2111	2066	TGGTCTCTTTTAGAGATTTACAGTGTTTTTCAACACTGTACGGTACC
	21	1	35	TGTAATACGACTCACTATAGGGTGGTCTCTTTTAGAGATTTACAGTGTTTTTCAACACTGTACG
mut-RNA3	22	1	51	TGTTAATACGACTCACTATAGGAAAAATACCAACTAATTCTCGTTCGATTCCGGCGAACATTCTATTTTACC	To generate mut-RNA3 template carrying the 5′ A → U substitution (underlined) at position 2 (Hema and Kao, 2004).
	23	2111	2066	GGCCTTAAGTGGTCTCTTTTAGAGATTTACAGTGTTTTTCAACACTGTACGGTACC

^aSee Materials and methods for detailed description of each construct.

^bRefers to the exact nt positions on the RNA3 template.

^cAll restriction marker sites introduced by primers are underlined, T7 promoter sequence is in bold.