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GeneCorner plasmid details

Last data update: 21 September 2022 10:23 CEST

Plasmid name: pROSA26-DV1 (LMBP 6451)

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Price category: Cat. 2 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p6451.gb (View with Genome Compiler)
p6451.txt
p6451.pdf
Sequence
analysis results
Genecorner:

NGS: c04-gc-dec2018-partial.fasta

Cloned DNA: B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP)
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:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Mouse phosphoglycerate kinase 1 polyadenylation signal (PGK1 polyA)
Bovine growth hormone polyadenylation signal (BGH polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Chloramphenicol (cam)
Neomycin (neo; G418)
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)
Mammalian cells
Parental clone: pBigT; pROSA26-1
Further information: The plasmid was constructed by subcloning the attR1-chloramphenicol resistance gene-ccdB-attR2 cassette as an EcoRV fragment into a blunt-ended XhoI site of the modified pBigT vector, downstream from the splice acceptor-loxP-PGK-neo-3xpA-loxP cassette and upstream of an IRES-EGFP-pA+ cassette. This plasmid was subsequently digested with the unique cutters AscI and PacI. The resulting fragment was cloned into the AscI/PacI opened pROSA26-1 vector.
pROSA26-DV1 is a monosite Gateway destination vector, containing the ccdB gene inserted between the attenuator sites R1 and R2. Next to this plasmid, an entry clone, containing the gene of interest flanked by the λ attL recombination sites, is needed in the monosite approach to target the gene of interest to the endogeneous ROSA26 locus in mouse ES cells. In this approach, the endogeneous ROSA26 promoter is used to drive transgene expression.
Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA.The G4 ROSALUC ES cells (LMBP 10507CB) are available at BCCM/GeneCorner as well.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726830.1.
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 attR1 and attR2 regions was analysed at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium).
The nucleotide sequence of the EGFP DNA corresponds with the EMBL Nucleotide Sequence Database accession number U55761.1.
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: U55761.1, view at EMBL, GenBank, DDBJ
HM588138.1, view at EMBL, GenBank, DDBJ
LT726830.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 01/08/2013
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: Alw44I/SacI, MluI/NdeI, NcoI, NheI/ScaI and NotI.

This plasmid has also been fully sequenced. The NGS sequence data still need to be implemented but were unfortunately incomplete.
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 first publication making use of 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 06350

Refer in your Materials and Methods:

pROSA26-DV1 (LMBP 6451) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr Jody Haigh and was published in Nyabi et al., 2009.

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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