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| Plasmid Name | Proper Citation | Insert Name | Organism | Bacterial Resistance | Defining Citation |
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pCAG-T3-hCAS-pA Resource Report Resource Website 10+ mentions |
RRID:Addgene_48625 | Codon optimized Cas9 | Synthetic | Ampicillin | PMID:23997119 | 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. | Vector Backbone:pCAGGS; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin | Addgene | 2026-10-10 01:54:09 | 20 |
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