Last data update: 24 January 2024 16:39 CET
Plasmid name: pSpCas9n(BB)-2A-GFP-GUImMLKL1 (LMBP 9363)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p9363.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Guide RNA targeting mouse mixed lineage kinase domain like (Mlkl, GeneID 74568) Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP) FLAG epitope tag; N-terminal SV40 large T-antigen nuclear localization signal (NLS); N-terminal Streptococcus pyogenes CRISPR associated protein 9 cDNA (Cas9); nickase mutant (Cas9n) Thosea asigna virus 2A peptide (T2A) |
| Promoter: | Human U6 small nuclear RNA gene promoter Chicken β-actin/rabbit β-globin hybrid promoter (AG) Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only |
| Ribosome binding site: |
- |
| Terminator: | Bovine growth hormone polyadenylation signal (BGH polyA) |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin Bacteriophage M13 origin |
| Host range: | Escherichia coli Mammalian cells |
| Parental clone: | pSpCas9n(BB)-2A-GFP (PX461) |
| Further information: | The plasmid was constructed by cloning the guide RNA sequence 1 for mouse Mlkl into the pSpCAS9n(BB)-2A-GFP (PX461) vector. This vector allows for genomic modification in the mouse Mlkl coding sequence via the CRISPR-Cas9 system. An aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain of the humanised S. pyogenes Cas9 coding sequence allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR22 can potentially decrease the frequency of unwanted insert/deletion mutations from off-target double-stranded breaks. Other name of the plasmid is pSpCAS9n(BB) - 2A - GFP - GUImMLKL1. |
| EMBL Accession number: | - |
| Latest sequence update: | 07/08/2017 |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | The plasmid was deposited by Prof. Dr M. Bertrand(1)(2). (1) Center for Inflammation Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Related plasmid reference: | Ran et al., Nat. Protoc. 8 (2013), 2281-2308 [PMID: 24157548] [DOI: 10.1038/nprot.2013.143] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 DH5α |
| Host reference: | Focus 8 (1986), 9 |
| Related host reference: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSpCas9n(BB)-2A-GFP-GUImMLKL1 (LMBP 9363) is available at BCCM/GeneCorner. The plasmid was deposited by Prof. Dr M. Bertrand. |
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.