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GeneCorner plasmid details

Last data update: 22 October 2020 04:25 CEST

Plasmid name: pCAGGS-E-hCYLD-R324A (LMBP 6645)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p6645.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human CYLD lysine 63 deubiquitinase cDNA (CYLD, CYLD1, USPL2, GeneID 1540); mutated sequence
E-tag; N-terminal
Promoter: Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
Ribosome
binding site:
-
Terminator: Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: pCD-F-hCYLD-R324A (FMSR/A); pCAGGSEhA20
Further information: The plasmid was created by PCR amplifying the C-terminal fragment of hCYLD using pCD-F-hCYLD-R324A (FMSR/A) as a template, and ligating it as a NotI/XhoI fragment in the NotI/XhoI opened pCAGGSEhA20 vector, replacing the hA20 cds.
The hCYLD cds contains a R324A mutation. The plasmid is used to test the MALT1 cleavage site. The arginine in position 324 was the only MALT1 target confirmed in both HEK293T cells and TNT in vitro translation/in vitro cleavage tests.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of the wild type human CYLD cDNA corresponds with the EMBL Nucleotide Sequence database accession numberAJ250014.1.
Other name of the plasmid is pCAGGS-E-CYLD-R324A.
EMBL Accession number: AJ250014.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 18/03/2010
Sequence detail:
Primers used to amplify the hCYLD cds:

Forward: 5' TCGTGCGGCCGCA.ATG.AGT.TCA.GGC.TTA.TGG.AGC 
                NotI      ***

Reverse: 5' GGCCTCGAG.TTA.TTT.GTA.CAA.ACT.CAT.TGT.TGG.A 
               XhoI   +++

***: start codon.
+++: termination codon.
Punctuation indicates reading frame.
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr J. Staal(1) (2) and Prof. Dr R. Beyaert(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Staal et al., EMBO J. 30 (2011), 1742-1752 [PMID: 21448133] [DOI: 10.1038/emboj.2011.85]
Related plasmid reference: Afonina et al., EMBO Rep. 17 (2016), 914-927 [PMID: 27113748] [DOI: 10.15252/embr.201642109]
Staal et al. (2016) [DOI: dx.doi.org/10.1101/046789]
Staal et al., bioRxiv (2016) [DOI: 10.1101/046789]
Restricted distribution: - BCCM MTA
- Possible extra restrictions defined by the depositor, based on the description of the enduser's research
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061
Host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Related host reference: Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

Refer in your Materials and Methods:

pCAGGS-E-hCYLD-R324A (LMBP 6645) is available at BCCM/GeneCorner. This plasmid was deposited by Dr J. Staal and Prof. Dr R. Beyaert and was published in Staal et al., 2011.

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.

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