Last data update: 24 January 2024 16:39 CET
Plasmid name: pT2-EF1a-hHOIL1-R165[P2A]-Blast (LMBP 9674)
| 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 RANBP2-type and C3HC4-type zinc finger containing 1 cDNA (RBCK1, HOIL1, RBCK2, RNF54, UBCE7IP3, XAP4, ZRANB4, GeneID 10616); mutated sequence Porcine teschovirus-1 2A peptide (P2A) Modified hyperactive sleeping beauty transposon arms |
| Promoter: | Human elongation factor 1α promoter (EF1α) Phage T3 promoter Phage T7 gene 10 promoter (T7g10) Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Bovine growth hormone polyadenylation signal (BGH polyA) |
| Selection marker: | Blasticidin (bsd) Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin Phage f1 origin |
| Host range: | Escherichia coli Mammalian cells |
| Parental clone: | pENTR-P4P1R-EF1a; pENTR221-hHOIL1-R165[P2A]; pENTR-C-BGH-Blast; pdT2-BH-L4L3 |
| Further information: | The plasmid was constructed via multisite Gateway LR recombination, combining the human EF1a promoter from pENTR-P4P1R-EF1a, the human RBCK1 mutant coding sequence linked to a P2A site from pENTR221-hHOIL1-R165[P2A] and the BGH terminator with blasticidin resistance gene from pENTR-C-BGH-Blast into the pdT2-BH-L4L3 vector. The human RBCK1 expression cassette is flanked by modified hyperactive sleeping beauty transposon arms consisting of IR/DRs (= inverted repeats containing short direct repeats) and the R165 residue in the MALT1 cleavage site of human RBCK1 was replaced by a P2A sequence, resulting in the expression of a constitutively cleaved protein. P2A is a self-cleaving peptide, fulfilling the same role as an IRES, but has a much shorter sequence. The purpose of this transposon plasmid is simple stable integration of the mutated human RBCK1 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. Other name of the plasmid is pT2-EF1a-hHOIL1-R165[P2A]-Blast. |
| 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-EF1a-hHOIL1-R165[P2A]-Blast (LMBP 9674) 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.