observed when the splicing cassette is inserted between other amino acid pairs within these coding sequences. Determination of the minimal gene context requirements for the P5SM-splicing cassette expands its general utility Expanding the repertoire of gene contexts beyond the wild-type E/R sequence would enable more facile design of splicing-regulated transgenes. Thus, to deﬁne the minimal context required for cassette function, we analyzed the effects of systematically mutating the border- ing codons. Mutations upstream of the 50-ss at positions 1 to 3 and downstream of the 30-ss at positions +1 to +3 were performed on the TFIIIA-eGFP reporter (Figure 2A). Since the amino acid changes are not in the Nucleic Acids Research, 2012, Vol. 40, No. 10 4705 eGFP sequence, the changes in ﬂuorescence reﬂect the activity of the splicing cassette. The effect of mutations to the bordering codons on splicing was analyzed by RT–PCR and on gene expression was quantiﬁed by ﬂuorescence leaf scanning (Figure 2B–D and Supplementary Figure S2). The results are consistent with the reported conservation pattern for nucleotides proximal to annotated 50-ss in A. thaliana (28), which we took to reﬂect the extent of inﬂuence on splice site strength. Position 3 had not been found to be highly conserved, and accordingly, the M1 and M2 constructs appear to retain full splicing activity. These mutants were characterized as splicing to SP-I and SP-II only, responding to AtL5 by an increase in SP-I, and ex- hibiting a similar ﬂuorescence induction to WT. The U Figure 2. Splicing activity of the cassette tolerates many, but not all, mutations to the bordering codons. (A) Schematic representation of the TFIIIA-eGFP gene construct showing the location of the mutated bordering codons (labeled NNN). The eGFP sequence is shown in green. Aberrant spliced products (red lines) are described in the text and the sequences are shown in Supplementary Figure S6. (B, C) Comparison of spliced products detected by RT–PCR for select 50-ss and 30-ss mutations tested within the TFIIIA-eGFP context (also see Supplementary Figure S2). Mutants were coexpressed with AtL5 (+) or LUC as a control ( ). Spliced products are labeled and the sequences are shown in Supplementary Figure S6. The mutants are categorized as fully functional (green), semi-functional (yellow) or non-functional (red). Red nucleotides indicate the mutations made to the bordering codon, and the corresponding encoded amino acid is listed below. (D) Fold induction of protein expression quantiﬁed by eGFP ﬂuorescence for select TFIIIA-eGFP mutants (also see Supplementary Figure S2). Induction with AtL5 was measured by comparison to induction with LUC as a control. For each mutant construct, the two amino acids encoded by the 50- and 30-bordering codons are indicated.