Stratagene California

Agilent Technologies, Inc.

Carsten-Peter Carstens

Joseph A Sorge

Jeffrey Carl Braman

Lee Scott Basehore

Natalia Novoradovskaya

Rajesh Bagga

Alan L Greener

John C Bauer

Henry Ji

Richard Gibbs

The present invention provides a method for making a large nucleic acid having a defined sequence in vivo. The method combines recombineering techniques with a CRISPR/Cas system to permit multiple insertions of defined sequences into a target nucleic acid at one time, double stranded cleavage of target nucleic acids in which the defined sequences were not successfully inserted, and selection of successful recombinant cells. The method further includes repeating the process one or more times, using a successful recombinant from one round as the host cell for the next round.

Methods of in vivo engineering of large sequences using multiple CRISPR/cas selections of recombineering events

The present invention relates to an isothermal method for detecting in a sample a target nucleic acid strand.

CAS9-based isothermal method of detection of specific DNA sequence

The invention provides cells and methods of circularizing linear DNA molecules. The cell is an isolatedcell which transiently expresses the Cre recombinase protein from an integrated Cre recombinase gene, and which is at least transiently repressed for RecBCD activity. The cells are used in a method of circularizing a linear DNA molecule comprising at least two loxP sites. The DNA molecule is introduced into the cells, and the linear DNA molecule is joined at said loxp sites.

Method for ligating nucleic acids and molecular cloning

The invention provides for polynucleotides and vectors comprising at least two tag sequences. The invention also provides for polynucleotides and vectors comprising a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for polynucleotides and vectors wherein a gene of interest is fused in frame to at least two tag sequences, for example, a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for the chimeric proteins encoded by these polynucleotides. The invention also provides for methods of using the polynucleotides of the invention for detecting and/isolating protein complexes or identifying a binding partner for a protein of interest.

Compositions and methods for protein isolation

The invention provides for polynucleotides and vectors comprising at least two tag sequences. In particular, preferred vectors are viral vectors. The invention also provides for polynucleotides and vectors comprising a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for polynucleotides and vectors wherein a gene of interest is fused in frame to at least two tag sequences, for example, a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for methods of using the polynucleotides and vectors of the invention for detecting and/isolating protein complexes or identifying a binding partner for a protein of interest.

The invention provides methods of covalently joining nucleic acid molecules and methods of molecular cloning. The methods provide either sequential or simultaneous ligation of flanking or vector nucleic acid molecules to nucleic acid insert molecules by topoisomerase and DNA ligase. The methods provide for directional and non-directional covalent joining and cloning of nucleic acid molecules.

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La Jolla, CA, United States of America

Carsten-Peter Carstens