Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg

Table 1 List of primers used for LMVmchVPg_Ec cloning and RT-PCR amplification

Primer name	Primer size (nt)	Amplified fragment	Sequence 5′-3′	Product size (kbp)
F1.fwd	30	F1	GCGGATCCAAAGGATAATATCATCACAGAG	8.5
F1.rev	28		GTATTGAACCATACGGTGCGTGATGGAG
F2.fwd	49	F2	*GACGAAGTATACCACCAGTCCGGA* GACGTCGCACGAAACTTCTGGAACG	7.7
F2.rev	23		CTTCTTCATCTGCCCAGAACCAC
mchV.fwd	58	mchV	CTCCATCACGCACCGTATGGTTCAATACAGTGATGTAGTGAGCAAGGGCGAGGAGGAT	1.3
mchV.rev	52		TGCGACGTCTCCGGACTGGTGGTATACTTCGTCTTCGTGCTTTACAGGGATG
P1Hc.fwd	20	3′P1-mchVPg-5′HcPro	GGCGACACAGTATATTCGTT	1.9
P1Hc.rev	21		TCACTAAAGTCATTCAGGAAC

The regions of homology allowing in vitro recombination between fragments are indicated in bold. The artificial NIa cleavage site is indicated in italics. The primers P1Hc.fwd and P1Hc.rev were used to perform RT-PCR to confirm the presence of the mcherry-VPg gene expression in the viral progeny. The suffixes “fwd” indicate sense primers, while suffixes “rev” indicated antisense primers. nt nuclotides, bp base pair

ISSN: 1743-422X