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To address this possibility, we inserted the region encompassing ACS2 in a YIp integrative vector that was then used to transform yeast cells.
GST-Axin-GBD (GSK-3 Binding Domain) fusion protein was constructed by inserting the region encoding residues 383 402 of human Axin into a modified pGEX-KG vector containing a TEV cleavage site between the GST protein and the insert.
UbiB also contains a 70 90 residue insert in the region corresponding to the activation loop in EPKs, but the function of this insert in UbiB is unknown.
This allowed the precise location of each insert within the region to be recorded.
Individual colonies were screened for the insert and the region of interest was sequenced using M13 primers.
In P. chrysosporium another insert in the region downstream of HD1 was found in two other individuals.
The Hydra protein has one amino acid deleted in this region, whereas the Amphimedon CBFβ domain shows a 2-residue insert the same region.
Indeed, the mutation G346E was inserted in the region of CCTα coding region that have been removed when constructing the KO heterokaryons strains.
The resulting DNA fragment was inserted into the 5' region of pGL3 basic promoter (Promega) to produce the wild type reporter construct.
Reno et al. (2009) also found that much of the variation among these genomes was caused by mobile elements inserted in the region we have termed N1.
In addition, when a selectable marker is inserted into the region instead of the PEN cluster, this transgene is not correctly expressed.
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