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The transgenes were rendered resistant to an RNAi construct that could effectively deplete endogenous CYK-4 by targeting the 3′ UTR and portions of the coding sequence.
We also demonstrated that the differences between the CS and ICCS vary along the transcript (Supplementary Fig. S4), although the background composition cannot explain the observed codon bias in all three portions of the coding sequence that were analysed individually.
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Due to this strategy, we expected to recover the 5' (and possibly the 3') UTR and some portion of the coding sequence.
A small portion of the coding sequence is annotated as an intron on the JGI Selaginella moellendorffii v1.0 genome site.
DAG1-null allele resulted in a deletion in the exon including the 3′ splice acceptor site and a large portion of the coding sequence of α-DG.
In each case, the indicated defect is predicted to interrupt a significant portion of the coding sequence required for function in well-characterized homologs.
MtCBF11 has most likely arisen from an in-frame fusion of the 5′ portion of the coding region from a first CBF/ DREB1 gene with the 3′ portion of the coding sequence from a second gene.
Inclusion of an intron in the 5' region of a gene, either in the 5'UTR or fused to the 5' portion of the coding sequence, leads to enhanced RNA levels [ 11- 15].
This may be due in part to an underrepresentation of annual BTC1 alleles in our panel or the fact that the current analysis was limited to a relatively small portion of the coding sequence of BTC1 (15%; Table 1), and did not include the promoter.
The double-stranded PCR products, which contained a portion of the coding sequence for the protein to be depleted flanked by T7 polymerase binding sites, were purified using a commercial kit (QIAquick Gel Extraction kit; Qiagen, Valencia CA, USA) and then used as template for RNA synthesis using a T7 RNA polymerase (Megascript T7 kit; Ambion, Austin TX, USA).
BLAST searching the coding DNA sequences of TaSUT2A, TaSUT2B and TaSUT2D against the IWGSC Survey Sequence Repository revealed contigs derived from the A, B and D genomes of wheat that contain a portion of the coding sequence (with over 99% sequence identity) and the respective 3′ UTR region of each homeologue.
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