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GeneCorner plasmid details

Last data update: 25 April 2019 04:11 CEST  


Name: pROSA26-DV2   Add to cart
Accession number: LMBP 6452
Sequence file: LMBP sequence: p6452.gb, p6452.txt     View with Genome Compiler
Circular map: p6452.pdf
Status: GeneCorner core plasmid
Cloned DNA: B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
Mouse gene trap ROSA 26 gene (Gtrosa26, Gt(ROSA)26Sor, beta geo, Gtrgeo26, R26, ROSA26, Thumpd3as1, GeneID 14910); 5' UTR and 3' UTR
Promoter: Mouse phosphoglycerate kinase 1 promoter (PGK1)
Phage T3 promoter
Phage T7 gene 10 promoter (T7g10)
Escherichia coli lac operon promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
Ribosome
binding site:
-
Terminator: Bovine growth hormone polyadenylation signal (BGH polyA)
Selection marker: Ampicillin (amp)
Chloramphenicol (cam)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli; use a ccdB-resistant strain for propagation (the Invitrogen One Shot ccdB Survival 2 T1R Competent Cells are not recommended for this plasmid)
Parental clone: pBigT-R4-ccdB-CmR-R3; pROSA26-1
Further information: The plasmid was created by blunt-end cloning the attR4-ccdB-chloramphenicol resistance gene-attR3 cassette from a pBigT-R4-ccdB-CmR-R3 plasmid into the PacI opened and blunted pROSA26-1 vector.
pROSA26-DV2 differs from pROSA26-DV3 only in the orientation of the Gateway cassette and the absence of the BGH polyA site between the Gateway cassette and the 3' UTR of ROSA26.
The plasmid is a multisite Gateway destination vector, containing the ccdB gene inserted between the attenuator sites R3 and R4. Next to this plasmid, three entry clones are needed in the multisite approach to target the gene of interest to the endogeneous ROSA26 locus in mouse ES cells: 1) a 5' entry clone with attL4-R1 sites and containing the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the loxP flanked β-galactosidase-neomycin fusion gene (β-geo) polyA termination cassette (e.g. pEntry attL4pCAGG-loxP-Bgeo-3xpA-loxP-attR1 (LMBP 6455)); 2) a middle entry clone with attL1-L2 sites and containing the gene of interest and 3) a 3' entry clone with attR2-L3 sites and containing the IRES-EGFP reporter (e.g. pEntry attR2-IRES-eGFP-luc+-pA-attL3 (LMBP 8200)). In this approach, a strong, exogeneous promoter is used to drive transgene expression, resulting in higher expression levels as compared to the monosite approach using the endogeneous ROSA26 promoter (see pROSA26-DV1 (LMBP 6451)). Compared to the ROSA26 promoter driven expression, the AG-based expression may become mosaic in various tissues.
With pROSA26-DV2, the AG promoter is inserted into the ROSA26 locus in the antisense orientation, which may be better then the sense orientation when using pROSA26-DV3 (LMBP 6453), at least in undifferentiated ES cells.
Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA.
The depositor suggests that the products of the LR reactions using this vector should be sequenced at their junctions as is outlined in Nyabi et al. (2009) to ensure correct plasmid recombination before final electroporation of the targeting constructs into ES cells.
The nucleotide sequence of the region from nucleotide position 11173 to 11695 and from nucleotide position 1 to 1763, spanning part of the 5' UTR region of ROSA26, the entire attR cassette and part of the 3' UTR region of ROSA26, was analysed at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium).
The nucleotide sequence of the 5' and 3' UTR regions of ROSA26 correspond with the EMBL Nucleotide Sequence Database accession number HM588138.1.
EMBL Accession number: HM588138.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 01/08/2013
Sequence detail:
Primers used to sequence the region described in 'Further information':

Forward primer: AAA GTC GCT CTG AGT TGT TAT

Reverse primer: GGA GCG GGA GAA ATG GAT ATG
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: AvaI/StuI, BglII/EcoRV, NcoI/SmaI, NheI/PvuII and ScaI.

This plasmid has also been fully sequenced but the NGS sequence data still needs to be implemented.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr Jody Haigh(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Nyabi et al., Nucleic Acids Res. 37 (2009), e55 [PMID: 19279185] [DOI: 10.1093/nar/gkp112]
Related plasmid reference: Soriano, Nat. Genet. 21 (1999), 70-71 [PMID: 9916792]
Restricted distribution: - VIB/BCCM MTA
- When appropriate in accordance with academic customs, RECIPIENT agrees to include the depositor(s) as co-author(s) in the initial publication describing the MATERIAL.
Distributed as: plasmid DNA
Host for distribution: Escherichia coli K12xB DB3.1
Host reference: -
Related host reference: Bernard et al., J. Mol. Biol. 226 (1992), 735-745 [PMID: 1324324]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml) + chloramphenicol (34 μg/ml)*
Cultivation temperature: 28°C
Biosafety level: L1
Cultivation remark: *: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin. The plasmid-carrying strain must be cultivated at 28°C instead of 37°C, under heavy shaking, to prevent recombination. After each cultivation, the plasmid has to be checked for recombination. Because of the size of the plasmid, use electro-competent bacterial strains for propagation.
Other culture collection numbers: LMBP 06351

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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