p35SPPDK-EGFP-Flag Plasmid

p35SPPDK-EGFP-Flag is a plasmid developed for the expression of EGFP (enhanced green fluorescent protein) in plant protoplasts. It can be used as positive control for protoplast transfection or be used to construct the EGFP fusion proteins to examine the subcellular localization of target proteins in protoplasts. There are multiple restriction enzyme digestion sites at the N-terminus of EGFP sequence and the fusion protein containing EGFP tag at the C-terminus can be obtained by inserting the target gene at the mutiple cloning sites. The C-terminus of EGFP sequence has 2X Flag tag (DYKDDDDKDYKDDDDK) which can be used to examine the expression level of the fusion proteins in protoplasts by Western blot. This plasmid contains the ampicillin resistant gene for bacterial selection.

The expression of the EGFP gene in the p35SPPDK-EGFP-Flag vector is driven by the 35SPPDK strong promoter which is a fusion of the cauliflower leaf virus 35S (CaMV35S) promoter and the maize C4PPDK promoter and can efficiently drive the expression of target proteins in plant cells.

The p35SPPDK-EGFP-Flag plasmid contains only the components required for transient expression in plants to ensure higher transformation efficiency of protoplasts. This vector cannot be used to obtain stable transgenic plants.

The p35SPPDK-EGFP-Flag plasmid employs the nopaline synthase terminator (NOS-T) from Agrobacterium tumefaciens to terminate the transcription by RNA polymerase and regulate the expression of target genes.

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