Last data update: 24 January 2024 16:39 CET
Plasmid name: pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1 (LMBP 8212)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p8212.gb
(View with Genome Compiler) p8212.txt p8212.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Reverse tetracycline-responsive transcriptional activator (rtTA) |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE); minimal promoter with tet operator (CMVT) |
| Ribosome binding site: |
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene |
| Terminator: | Bovine growth hormone polyadenylation signal (BGH polyA) Escherichia coli rrnB operon T1 terminator Escherichia coli rrnB operon T2 terminator |
| Selection marker: | Neomycin (neo; kanamycin (kan)) Puromycin (puro) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli Mammalian cells |
| Parental clone: | pENTRY-L4-R1 |
| Further information: | The plasmid is a multisite Gateway entry vector, designed as a 5' entry clone (with attL4-attR1 sites) for the multisite approach using the pCOIN DV vector. The plasmid contains: - a reverse tetracyclin transactivator (rtTA) - an IRES-puromycin-pA drug-selectable cassette to select for rtTA expressing cells after Cre excision of the pCOIN DV vector. - a tetracycline responsive element (TRE), consisting of the minimal human CMV promoter, combined with the tet operator. - two adjacent copies of the chickenβ-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator core sequence. Insulators are DNA sequences which possess the ability to protect expressing genes from inappropriate signals emanating from their surrounding environment by acting as barriers that prevent the advance of nearby condensed chromatin that may otherwise silence expression. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726877.1. The nucleotide sequence of the rtTA corresponds with the EMBL Nucleotide Sequence Database accession number U89930.1. |
| EMBL Accession number: | U89930.1, view at EMBL, GenBank, DDBJ LT726877.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 08/07/2013 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: ApaI, HindIII, PvuI, PvuII and SacII. As compared to the compiled nucleotide sequence, the restriction enzyme pattern revealed that the plasmid is about 150 to 200 bp shorter. This fragment is probably located between the Tn903 neomycin resistance and the pMB1 ori. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr L. Haenebalcke(1) (2) and Prof. Dr J. Haigh(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Haenebalcke et al., Stem Cell Rev. Rep. 9 (2013), 774-785 [PMID: 23877658] [DOI: 10.1007/s12015-013-9458-z] |
| Related plasmid reference: | PhD thesis Lieven Haenebalcke |
| Restricted distribution: | - VIB/BCCM MTA - The depositor will be informed of the customer's identity and of the enduser's research described in the VIB/BCCM MTA upon release of a sample outside the depositor's department. - When appropriate in accordance with academic customs, RECIPIENT agrees to include the depositor(s) as co-author(s) in the first publication making use of the MATERIAL. |
| 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 + kanamycin (50 μg/ml) |
| Cultivation temperature: | 28°C |
| Biosafety level: | L1 |
| Cultivation remark: | This culture grows very slow: take 24 hours into account for saturated growth! |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1 (LMBP 8212) is available at BCCM/GeneCorner. This plasmid was deposited by Dr L. Haenebalcke and Prof. Dr J. Haigh and was published in Haenebalcke et al., 2013. |
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.