id	author	title	date	pages	extension	mime	words	sentences	flesch	summary	cache	txt
cord-293966-5c466xvz	Fehr, Anthony R.	Bacterial Artificial Chromosome-Based Lambda Red Recombination with the I-SceI Homing Endonuclease for Genetic Alteration of MERS-CoV	2019-09-14		.txt	text/plain	3813	229	62	Quickly after the identification of Middle East respiratory syndrome-CoV (MERS-CoV), both in vitro ligation and BAC-based reverse genetic technologies were engineered for MERS-CoV to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. Here, I will describe how lambda red recombination can be used with the MERS-CoV BAC to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the MERS-CoV genome and recover recombinant virus. In the method described here, this recognition site is engineered on a plasmid (pEP-KanS) just outside of the positive selection marker, and its cleavage with the I-SceI enzyme allows for the removal of the positive selection marker by intramolecular Red recombination utilizing sequence duplication introduced in the original PCR primers. Using 1 μL of the BAC DNA, use the external primers located 100-200 bp outside the region of homology you previously designed to test for the insertion of Kan r -I-SceI by PCR.	./cache/cord-293966-5c466xvz.txt	./txt/cord-293966-5c466xvz.txt