Last data update: 04 December 2020 04:17 CET
Plasmid name: pLenti6-V5-APEX blast (LMBP 9184)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
Glycine max cytosolic ascorbate peroxidase 1 cDNA (APX1, GeneID 553156); monomeric enhanced mutant (APEX), C-terminal
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)
Synthetic prokaryotic EM7 promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|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)
|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-Flag blast|
|Further information:||The plasmid was constructed by cloning the V5 epitope and the APEX coding sequence into the pLenti6-Flag blast vector, replacing the Flag epitope.
APEX, the monomeric enhanced APX mutant, contains the three engineered mutations K41D, W41F and E112K and is used as a reporter to generate contrast for electron microscopy and also for proteomic tagging in living cells.
This is a lentiviral Gateway destination vector, suitable for microRNA expression.
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.
Depending on the gene cloned in this vector, some leakage expression may be detected.
Because of the high risk for recombination, plasmid integrity should be checked via PvuII restriction digest after each subcultivation.
This Gateway destination vector is compatible with the human orfeome library which is also available at BCCM/GeneCorner.
This vector can not be used to express short hairpin RNA (shRNA), which requires an RNA-polymerase III promotor. However, this vector can be used for knock-down experiments if the hairpin is expressed in a micro-RNA context (eg. Block-iT PolII miR RNAi kit).
The nucleotide sequence of the pLenti6-Flag blast vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009451.1.
Other name of the plasmid is pLenti6(bla) -V5 - APEX - dest.
|EMBL Accession number:||LT009451.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||07/08/2017|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||The plasmid was deposited by Prof. Dr M. Bertrand(1)(2).
(1) Center for Inflammation Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|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|
|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)*|
|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.