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URL: http://www.addgene.org/48625
Proper Citation: RRID:Addgene_48625
Insert Name: Codon optimized Cas9
Organism: Synthetic
Bacterial Resistance: Ampicillin
Defining Citation: PMID:23997119
Vector Backbone Description: Vector Backbone:pCAGGS; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Comments: SV40 nuclear localization signals (NLS) were added to the N- and C-terminus of Cas9 by PCR using NLS-coding primers. The NLS flanked Cas9 was inserted into the BglII-Acc65I site of ZFN-platform vector to add a T3 promoter, FLAG-tag and the Tbpl1 3′UTR containing a 95 bp polyA tail. The sequence from the T3 promoter to the polyA tail was cloned by PCR and inserted into the EcoRI site of the pCAGGS vector. For the in vitro synthesis of CAS9 mRNAs, the vector was linearized by SphI and in-vitro transcribed using T3-RNA-polymerase (Promega) in the presence of m7G(5′)ppp(5′)G to synthesize capped RNA. The Tbpl1 3′UTR 95 nucleotide polyA tail at the 3' end of the insert allows in vitro transcribed mRNA from this plasmid to be used directly for microinjection into animal embryos or oocytes without additional polyadenylation treatment. The CAG promoter in this plasmid also allows it to be used as a CAS9-expression vector.
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Source: Addgene (RRID:SCR_002037)
Description: Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
URL: http://www.addgene.org
The NIDDK Information Network (dkNET) serves the needs of basic and clinical investigators by providing seamless access to large pools of data and research resources relevant to the mission of The National Institute of Diabetes Digestive and Kidney Diseases (NIDDK). dkNET is hosted at University of California San Diego, and is supported by NIH NIDDK grant U24DK097771.
Email: info_at_ dknet.org
