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GeneCorner plasmid details

Last data update: 24 January 2024 16:39 CET

Plasmid name: pT2-UNO-E-hA20-R439[P2A]-Blast (LMBP 9611)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: not available
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human TNF alpha induced protein 3 cDNA (TNFAIP3, A20, OTUD7C, GeneID 7128); mutated sequence
E-tag; N-terminal
Porcine teschovirus-1 2A peptide (P2A)
Modified hyperactive sleeping beauty transposon arms
Promoter: Human elongation factor 1α/human T-cell leukemia virus type 1 long terminal repeat hybrid promoter (EF1/HTLV)
Phage T3 promoter
Phage T7 gene 10 promoter (T7g10)
Escherichia coli lac operon promoter
Simian virus 40 early promoter (SV40 early)
Ribosome
binding site:
-
Terminator: Bovine growth hormone polyadenylation signal (BGH polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Blasticidin (bsd)
Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: pENTR-P4P1R-UNO; pENTR221-E-hA20-R439[P2A]; pENTR-C-BGH-Blast; pdT2-BH-L4L3
Further information: The plasmid was constructed via multisite Gateway LR recombination, combining the EF1/HTLV promoter from pENTR-P4P1R-UNO, the mutated human TNFAIP3 coding sequence linked to the P2A sequence from pENTR221-E-hA20-R439[P2A] and the BGH terminator with blasticidin resistance gene from pENTR-C-BGH-Blast into the pdT2-BH-L4L3 vector.
The E-tagged human TNFAIP3 expression cassette is flanked by modified hyperactive sleeping beauty transposon arms consisting of IR/DRs (= inverted repeats containing short direct repeats) and the R439 residue in the MALT1 cleavage site of the human TNFAIP3 coding sequene was replaced by a P2A sequence, resulting in the expression of a constitutively cleaved protein.
The purpose of this transposon plasmid is simple stable integration of the E-tagged and mutated human TNFAIP3 coding sequence in a cell by co-transfection with a sleeping beauty transposase (SB100X). Sleeping Beauty (SB) transposon-based transfection is a two-component system consisting of a transposase and a transposon containing IR/DR sequences that result in precise integration into a TA-dinucleotide site.
The EF1/HTLV hybrid promoter is a composite promoter comprising the Elongation Factor-1α (EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity, higher than viral promoters and, on the contrary to the CMV promoter, yields persistent expression of the transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of DNA and RNA. This hybrid promoter was originally obtained from pUNO1.
EMBL Accession number: -
Latest sequence update: 28/02/2018
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. R. Beyaert(1) (2). It was constructed by J. Meert(2)(3).
(1) VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
(3) BCCM/GeneCorner Plasmid Collection, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
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:

pT2-UNO-E-hA20-R439[P2A]-Blast (LMBP 9611) is available at BCCM/GeneCorner. This plasmid was deposited by Dr J. Staal and Prof. 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.

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