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Fig. 1 | Virology Journal

Fig. 1

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

Fig. 1

Cloning of LMVmchVPg_Ec full-length cDNA by in vitro recombination Gibson assembly strategy. a Schematic representation of the infectious clone LMVmchVPg_Ec genome. The ectopically expressed mcherry-VPg fusion protein gene is inserted between the P1 and HcPro sequences of the Lettuce mosaic virus (LMV) genome (red star: mcherry fluorescence tag fused at the N-terminus of the VPg). b Schematic representation of the cloning by in vitro recombination strategy. The size of the overlapping regions between the PCR fragments where recombination takes place is indicated. The three overlapping fragments named mchV, F1 and F2 (highlighted in red, blue and green respectively) were amplified by long distance PCR using the primers pairs (arrows) positioned along the cloning vector or the viral LMV template. The full-length cDNA of LMV is derived from the LMV-0 cDNA clone previously reported by Redondo et al. [30] and further modified by Sorel et al. [23]. The LMV FL- cDNA was assembled into a pBluescribe-derived vector pBS70T containing an enhanced 35S promoter (P70S), a NOS terminator (NosT), an E.coli replication origin (Ori), an ampicillin resistance gene (AmpR), a cassette containing the 2 μ yeast replication origin and a yeast selectable marker (Trp-1promoter and gene, 2 μ-Trp1).The three PCR products were assembled in vitro by Gibson assembly. bp: base pair; kbp: kilobase pair

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