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The plasmid pPIC9K- hμPlg contained a 783-bp inserted target fragment.
As expected, we observed a specific knockdown that corresponded to the inserted target sequences.
In the inserted target gene template DNA, a SalI digestion site was designed between BamHI and HindIII of Pgenesil-1.
The inserted target segments in recombinant plasmids were sequenced using the double-stranded dideoxy chain termination method by Invitrogen Co., Ltd ,Shanghai, China.
Based on the structure of polyomavirus capsids [ 13, 14], it was predicted that the inserted target sequence can be displayed on the surface of pseudotype VLPs.
Each vector has a N-terminal fusion 'tag' (Table 1) followed by Rhinovirus 3C protease cleavage site between the 'tag' and the inserted target sequence to facilitate 'tag' removal.
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We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector.
Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus.
A guarded, 27-gauge 0.5-in needle was stereotactically inserted, targeting the lateral ventricle (3.5 mm depth).
However, to maximise the utility of tagged sequence mutagenesis, one would like to predict, from the sequence alone, the functional consequences of the inserted targeting vector on cellular gene expression.
One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants.
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