Figure 3. Schematic of splicing issues in the putative CDK12 analog sensitive cell line (clone #5, figure 1 and figure 2). Mutation of the TTT codon encoding F813 of CDK12 to GGT (F813G) results in the creation of a novel splice acceptor site downstream of the canonical one between exon5 and exon6 of CDK12. Consensus splicing sequences are shown at the upper right of the figure. A schematic of exon 5 (black text), the intron between exons 5 and 6 (gray lowercase text), and exon 6 (blue text) is depicted in the parental (WT) and putative analog sensitive cell line (AS). The PAM and analog sensitive mutations are highlighted in red. Note how mutation of F813G with TTT->GGT results in the formation of a “tgtag” sequence motif exactly the same as the motif present at the end of the intron. Chromatographs of TA cloned cDNA sequences from this site are shown below the mis-spliced sequence; the end of exon 5 is highlighted in blue. This unanticipated splicing results in an 6AA truncation of the CDK12 protein; the resultant protein retains residual activity, but is not analog-sensitive (data not shown).