Last data update: 24 October 2020 04:12 CEST
Plasmid name: pBlue-mA20f3 (LMBP 3952)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Mouse tumor necrosis factor, alpha-induced protein 3 cDNA (Tnfaip3, A20, Tnfip3, GeneID 21929); fragment
|Promoter:||Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter
|Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)|
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli; use strains expressing the lacZ ω fragment (lacZΔM15) for screening|
|Further information:||pBlue-mA20f3 was constructed as follows: 1) pBluescriptIIKS- was digested with BamHI, filled in with Klenow DNA polymerase and religated. 2) a 1116 bp KpnI-SalI PCR fragment, containing codons 1 up to and including 366 of the mouse zinc finger protein A20 coding sequence (mA20), was then inserted between the SalI and KpnI sites.
pBlue-mA20f3 contains the T7 and T3 RNA polymerase promoters for efficient in vitro RNA synthesis of the coding or non-coding DNA strand.
This plasmid also contains the replication origin of the single-stranded DNA phage f1 (clockwise orientated), so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage, e.g. M13KO7.
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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727290.1.
The nucleotide sequence of pBluescriptIIKS- corresponds with the EMBL Nucleotide Sequence Database accession number X52329.1.
The nucleotide sequence of the mA20 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number U19463.1 and adapted to the sequence analysis results of the Department of Biomedical Molecular Biology (Ghent University, Belgium). The following difference is observable: the nucleotide T at position 2096 is substituted by a C- residue, resulting in a Leu/Pro replacement.
Other names of the plasmid are pBluescript-mA20(1-366) and pB-001-006#1.
|EMBL Accession number:||U19463.1, view at EMBL, GenBank, DDBJ
LT727290.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||23/11/2001|
------ mA20 ------> Forward primer: 5' CGGGATCCGTGGTACCGCC.ATG.GCT.GAA.CAA.CTT 3' BamHI KpnI *** <------------ mA20 ------------ Reverse primer: 5' GAAGATCTTCGTCGACAC.TTA.CGC.TCT.CCT.GGC.CTG.ATC.GCT.GTT 3' BglII SalI +++ 366 ***: start codon. +++: termination codon. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: KpnI, NciI, NcoI/SalI and XmnI.|
|History of deposit:||This plasmid was deposited by 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
|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:||Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pBlue-mA20f3 (LMBP 3952) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert .|
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.