Last data update: 24 January 2024 16:39 CET
Plasmid name: pT2-UNO-E-hA20-R439A-Blast (LMBP 9612)
New search | Print data sheet |
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 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-R439A; 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 from pENTR221-E-hA20-R439A 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). The human TNFAIP3 coding sequence contains an R439A mutation. 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-R439A-Blast (LMBP 9612) 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.