Fig. 5 Plasmid and primer sequences at junctions of a dual expression cassette for co-expression of eGFP and
mCherry fused to a C-terminal histidine tag. The eGFP and mCherry cDNAs were cloned into the pKL derivative
pKL-PH-3C-10His-Cter. PCR primers were designed to generate DNA fragments with 19- to 21-bp-long
overhangs and maintain the NheI, XhoI, BamHI, and XbaI restriction sites. The 3
0
region of the eGFP cDNA is
detailed in panel (a), the dual promoter region with the 5
0
region of the eGFP cDNA, the p10 and PH promoters,
and the 5
0
region of the mCherry cDNA in panel (b), and the 3
0
region of the mCherry cDNA in panel (c). The
plasmid backbone is amplified with the oligonucleotide pair Backbone-3C-R/Backbone-F and the dualpromoter module with the Prom-R/Prom-F pair. The eGFP and mCherry cDNAs are amplified with the p10gene-START-F/p10-gene-STOP-R and the pH-gene-START-F/pH-gene-10His-3C-R. The amino acid sequence
recognized by the prescission protease (3C) is LEVLFQ!GP (see Table 2)
mCherry fused to a C-terminal histidine tag. The eGFP and mCherry cDNAs were cloned into the pKL derivative
pKL-PH-3C-10His-Cter. PCR primers were designed to generate DNA fragments with 19- to 21-bp-long
overhangs and maintain the NheI, XhoI, BamHI, and XbaI restriction sites. The 3
0
region of the eGFP cDNA is
detailed in panel (a), the dual promoter region with the 5
0
region of the eGFP cDNA, the p10 and PH promoters,
and the 5
0
region of the mCherry cDNA in panel (b), and the 3
0
region of the mCherry cDNA in panel (c). The
plasmid backbone is amplified with the oligonucleotide pair Backbone-3C-R/Backbone-F and the dualpromoter module with the Prom-R/Prom-F pair. The eGFP and mCherry cDNAs are amplified with the p10gene-START-F/p10-gene-STOP-R and the pH-gene-START-F/pH-gene-10His-3C-R. The amino acid sequence
recognized by the prescission protease (3C) is LEVLFQ!GP (see Table 2)