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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLenti6(puro)-EYFP (LMBP 6783)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p6783.gb (View with Genome Compiler)
p6783.txt
p6783.pdf
Sequence
analysis results
Genecorner:

-

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 yellow-green fluorescent variant (EYFP)
Human immunodeficiency virus pol gene; central polypurine tract (HIV-1 cPPT)
V5 epitope; C-terminal
Promoter: Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Escherichia coli lac operon promoter
Simian virus 40 early promoter (SV40 early)
Phage T3 promoter
Phage T7 gene 10 promoter (T7g10)
Ribosome
binding site:
-
Terminator: Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 3' LTR); modified HIV-1 3' LTR with deleted U3 region (ΔU3/3' LTR)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Chloramphenicol (cam)
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli; use a ccdB-resistant strain for propagation
Mammalian cells; SV40 permissive cells
Parental clone: pLenti6.2/V5-DEST; pDONR207
Further information: The plasmid was constructed as follows: 1) the blasticidin coding sequence from pLenti6.2/V5-DEST was replaced by the puromycin selection marker and the EYFP coding sequence was inserted N-terminal of the V5 epitope; 2) the resulting entry vector was used to create an expression vector via Gateway LR reaction; 3) the resulting expression vector was used to recreate an entry vector by inserting the ccdB-chloramphenicol cassette of pDONR207 via Gateway BP reaction. As a result, the orientation of the attR cassette in pLenti6(puro)-EYFP is inverted compared to the parental pLenti6.2/V5-DEST vector. The V5 epitope is not functional.
This is a lentiviral Gateway destination vector designed for fusing a gene of interest to the N-terminus of EYFP and for ViraPower II lentiviral-based expression of the target gene in dividing and non-dividing mammalian cells.
The ViraPowerLentiviral Expression System (Invitrogen) possesses features which enhance its biosafety while allowing high-level gene expression in a wider range of cell types than traditional retroviral systems.
The vector carries the human CMV-IE promoter for driving constitutive expression of the target gene. Although highly active in most mammalian cell lines, activity of the viral promoter can be down-regulated in some cell lines due to methylation, histone deacetylation, or both.
The U3 region of the HIV-1 3' LTR is deleted (ΔU3 or dU3) and facilitates self-inactivation of the HIV-1 5' LTR after transduction to enhance the biosafety of the vector. The element also contains a polyadenylation signal for transcription termination and polyadenylation of mRNA in transduced cells.
The vector contains the psi packaging signal, allowing viral packaging, and the rev response element (RRE) from HIV-1, permitting Rev-dependent nuclear export of unspliced viral mRNA.
Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA.
This Gateway destination vector is compatible with the human orfeome library which is also available at BCCM/GeneCorner.
Other name of the plasmid is pLenti6(puro)-EYFP cl2.
EMBL Accession number: -
Latest sequence update: 28/09/2010
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: NcoI and PstI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by K. Vandepoele(1) (2) and Prof. Dr F. Van Roy(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Curradi et al., Mol. Cell. Biol. 22 (2002), 3157-3173 [PMID: 11940673]
Dull et al., J. Virol. 72 (1998), 8463-8471 [PMID: 9765382]
Kjems et al., Proc. Natl. Acad. Sci. U.S.A. 88 (1991), 683-687 [PMID: 1992459]
Malim et al., Nature 338 (1989), 254-257 [PMID: 2784194]
Rietveld et al., EMBO J. 21 (2002), 1389-1397 [PMID: 11889044]
Restricted distribution: - BCCM MTA
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 (25 μg/ml)*
Cultivation temperature: 28°C
Biosafety level: L1 in E. coli; L2 in mammalian cells
Cultivation remark: *: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin.
Other culture collection numbers: -

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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