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LMBP plasmid details

Last data update: 23 September 2020 04:27 CEST

Plasmid name: pLenti(puro)-mKate2 (LMBP 8522)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: -
Sequence
analysis results
Genecorner:

-

Cloned DNA: Entacmaea quadricolor red fluorescent protein DNA (eqFP578, RFP); super bright monomeric far-red fluorescent next generation TagFP635 variant (mKate2)
V5 epitope; C-terminal
B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
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-V5 puro
Further information: This lentiviral Gateway destination vector was constructed by cloning the mKate2 coding sequence downstream from the Gateway cassette in pLenti6-V5 puro.
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 of the high risk for recombination, plasmid integrity should be checked via PvuII restriction digest after each subcultivation.
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.
The nucleotide sequence of the mKate2 coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number JN418198.1.
The nucleotide sequence of the pLenti6-V5 puro vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009457.1.
Other name of the plasmid is pLenti(puro) mKate2 cl3 bis.
EMBL Accession number: JN418198.1, view at EMBL, GenBank, DDBJ
LT009457.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 02/10/2013
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: AgeI, EcoRV, KpnI, NcoI, PvuI and XmaI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr F. van Roy(1) (2). It was constructed by E. Sanders(1) (2).
(1) VIB Center for Inflammation Research, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: De Groote et al., BioTechniques 60 (2016), 252-259 [PMID: 27177818] [DOI: 10.2144/000114417]
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)
Cultivation temperature: 28°C
Biosafety level: L1 in E. coli; L2 in mammalian cells
Cultivation remark: This culture grows very slow: take 24 hours into account for saturated growth! Take a very low plasmid yield (18-23 nanogram/microliter) into account while choosing a plasmid DNA isolation procedure.
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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