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. 2014 Aug 28;9(8):e105932. doi: 10.1371/journal.pone.0105932

Figure 4. Schematic diagram of synthesised constructs, with restriction enzyme sites incorporated for subsequent cloning.

Figure 4

A) pSPLITrep 1-219Rb-35S containing “modules” that could be removed and replaced with other sequences by restriction digest. B) Illustration showing how the rep 1-219Rb- transgene was split at the first AGGC (nucleotides 155, 156, 157 and 158 with respect to the start codon). The exon 2, cloned at the 5′ terminus of the split gene cassette in A) therefore began with GC, and the exon 1, cloned at the 3′ terminus, ended in AG. C) The synthesised rep III-Rb- (see Fig. 1A for the full-length gene product) exon 2, preceded by the 3′-terminal half of the syntron, flanked by SwaI and SpeI RE sites. The 3′-terminal syntron/rep 1-219Rb- exon 2 in pSPLITrep 1-219Rb-35S was replaced by the 3′-terminal syntron/rep III-Rb- exon 2 to create pSPLITrep III-Rb-35S. Exon 1 remained the same for both constructs since they share the same 5′-terminal 156 bp. Similarly, other modules were exchanged to create further constructs, such as the CaMV 35S promoter for the maize ubiquitin promoter etc (see text for details).