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Among them: falcon wing doors that wouldn't close and proximity sensors that failed to sense an overhang, allowing the doors to bang into it.
Furthermore, the localization of Cdc13 to the telomeres by the DBD domain would position Cdc13N in close proximity to the telomeric overhang, allowing for preferential binding of the telomeric DNA over pol α when long single stranded overhangs are available (i.e., late S phase/G2).
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Further enzymatic digestion to generate the second compatible overhang allows intramolecular ligation and plasmid circularisation.
The small amount of overhang allowed by h is necessary on the end of the overlapping portion to allow for poorly trimmed sequence.
The Type IIS sites were designed to result in cuts within the repetitive sequence and to produce compatible DNA overhangs allowing for seamless elongation of the Poly-Q encoding sequence.
A "double SfiI" strategy utilizing two non-complementary overhangs allows insert swapping to be carried out with a single, robust restriction/ligation cycle.
In addition, the use of a short double stranded DNA oligonucleotide with 3′ overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%.
The deposition process includes adsorption and migration processes and it is modeled via kinetic Monte Carlo simulation on a triangular lattice with vacancies and overhangs allowed to develop inside the film.
As shown in Supplemental Figure 7C&D BstXI-digested overhangs allow unidirectional cloning and prevent insertion of tandems of oligonucleotide duplexes.
The generated complementary overhangs allowed introduction of the target gene into the vectors treated with T4 DNA polymerase using a simple annealing step, without use of any other enzyme.
Introduced T-overhangs allowed the insertion of the fragments into pGEM-Teasy being spaced by the AF riboB sequence which was amplified from A. fumigatus genomic DNA using oligonucleotide combinations primer 5&6.
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