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. 2014 Jun 10;9(6):e99041. doi: 10.1371/journal.pone.0099041

Figure 2. Schematic structure of vector constructs encoding RCY1.

Figure 2

P35S-cRCY1-HA contains the RCY1 cDNA with no introns but including an HA-epitope tag (HA) at its 3′-end, the 66-bp 5′ sequence upstream of the RCY1 start codon of (66-bp 5′-UTR), and the 71-bp 3′-downstream sequence from the RCY1 stop codon (71-bp 3′-UTR). All elements were cloned between the CaMV 35S promoter (P35S) and the 5′-UTR sequence of the Arabidopsis Alcohol Dehydrogenase gene (ADH5′-UTR) and terminator sequence of the Heat Shock Protein gene (THSP) in the pRI201-AN binary vector. PRCY1-gRCY1-HA, contains the 1.5 kb genomic RCY1 promoter (PRCY1), the genomic RCY1-coding regions including two introns and tagged with HA at its 3′-end, and the RCY1 66-bp 5′-UTR region and the 71-bp 3′-UTR cloned into the pRI201-AN binary vector. P35S-gRCY1-HA::P35S-GFP and PRCY1-gRCY1-HA::P35S-GFP include P35S, the GFP-coding region, and the nos terminator (TNOS) cloned into the vector pRI201-AN. P35S-gRCY1-HA, includes the genomic RCY1-coding region with two introns tagged with HA at its 3′-end, and the RCY1 66-bp 5′-UTR and 71-bp 3′-UTR, cloned downstream of P35S between ADH5′-UTR and THSP in pRI201-AN. P35S-gRCY1-HAorf was constructed by deletion of 66-bp-5′-UTR and 71-bp-3′-UTR from P35S-gRCY1-HA. P35S-gRCY1.ΔI-HA and P35S-gRCY1.ΔII-HA were constructed by deletion of the first or second intron from P35S-gRCY1-HA, respectively. P35S-cRCY1.Ic-HA was constructed by insertion of the COLD-REGULATED 15A (COR15a) intron II into the first splice junction site in P35S-cRCY1-HA. P35S-cRCY1.Ip-HA was created by insertion of the PROFILIN 3 (PRF3) intron I into the first splice junction site in P35S-cRCY1-HA. In all vector constructs, the 66-bp 5′-UTR and 71-bp 3′-UTR of RCY1 are shown as black boxes. The RCY1-coding region is indicated by gray boxes in which the two splice junction sites are indicated by vertical lines in the boxes. The two RCY1 introns are shown as black dashed lines and the COR15a or PRF3 introns are shown as gray dashed lines.