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31	Design of gRNA and construction of gRNA expression vectors Originally developed by Hodaka Fujii on February 19, 2014 Modified by Hodaka Fujii on January 10, 2015 Construction of gRNA expression vectors is done basically Add to Reading List Source URL: www.addgene.org Language: English - Date: 2015-01-22 10:39:59 Guide RNA Vector Retrovirus Nucleosome Promoter Restriction enzyme Viral vector Gene expression Expression vector Biology Molecular biology Biochemistry
32	gRNA Synthesis Protocol STEP 1: Find all 23bp genomic sites of the form 5’-N20NGG-3’ near your intended target site (ideally ±50bp). These may reside on the + or – strand. PAM 5’!NNNNN NNNNN NNNNN NNNNN NGG!3’ Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:32:09 Chemistry Guide RNA Polymerase chain reaction Oligonucleotide Molecular cloning Vector DNA microarray Gene expression Biology Molecular biology Biochemistry
33	CRISPR plasmids from Chen and Wente labs We have synthesized a Cas9 coding sequence with nuclear localization signals (nls) at both its amino and carboxyl termini and codons optimized for zebrafish expression (nlszCas9-n Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:32:00 Genetics Molecular genetics DNA ligase DNA replication Guide RNA Nuclear localization sequence Transformation DNA Ligation Biology Molecular biology RNA
34	Generating targeting chiRNAs A. Selecting a target sequence The genomic target sequence should fulfill the following requirements: • 20-nt long • followed by a 3-nt PAM sequence: NGG • begin with a G to optimize U6 Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:31:56 Nucleic acids RNA DNA Oligonucleotide Sense Plasmid Biology Molecular biology Microbiology
35	Fire Lab C. elegans Vector Kit Description: The Andrew Fire laboratory has deposited a set of 288 vectors for C. elegans research. The plasmids are divided into three plates: 1995 plate, 1997 plate, and 1999 plate. 24 ve Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:32:07 Gene delivery Microbiology Mobile genetic elements Plasmid Vector Caenorhabditis elegans Biology Molecular biology Biochemistry
36	Advanced Gateway Destination Vectors Description: The Susan Lindquist laboratory of the Howard Hughes Medical Institute and the Whitehead Institute for Biomedical Research has deposited a set of Gateway® destination vec Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:32:02 Green fluorescent protein Chemistry Biology Christian Commission for Development in Bangladesh Christianity in Bangladesh
37	Open Source Wnt Kit Description: The Open Source Wnt Project was initiated to create a collection of standardized Wnt plasmids that are easily accessible to the research community. The project and plasmids were created b Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:31:57 Signal transduction Wnt signaling pathway WNT7B WNT3A WNT1 WNT3 WNT7A WNT4 Plasmid Biology Biochemistry Genes
38	Guide to Depositing Synthetic Biology Parts When depositing your standard parts with Addgene, we encourage you to include some additional information which will make it easier for requesting scientists to use them in fut Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-18 11:27:50 Microbiology Gene delivery Biotechnology Plasmid BioBrick IGEM Biology Synthetic biology Molecular biology
39	Myristoylated Kinase Library Description: This library consists of 192 human kinases and kinaserelated open reading frames (ORFs) cloned into a retroviral vector which adds a myristoylation sequence and flag-epitope tag Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:32:02 Gene delivery Microbiology Plasmid Vector Biology Molecular biology Biochemistry
40	Choice of gRNAs for the Cas9 Orthologs S. pyogenes Cas9 (SP): Find all 23bp genomic sites of the form 5’-NNNNNNNNNNNNNNNNNNNNNGG-3’ near your intended target site (ideally ±50bp). These may reside on the + or – st Add to Reading List Source URL: www.addgene.org Language: English - Date: 2014-11-05 23:31:59 Molecular biology Genetics DNA Gene expression RNA Biology Guide RNA

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