Last data update: 04 December 2020 04:17 CET
Plasmid name: pLenti6-Flag blast (LMBP 9163)
|Price category:||Cat. 3 (cf. price list)|
|Status:||GeneCorner core plasmid|
|GeneCorner sequence:||p9163.gb (View with Genome Compiler)|
|Cloned DNA:||B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
FLAG epitope tag; 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
|Further information:||The construction of this plasmid is described in De Groote et al. (2016).
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.
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 corresponded with the EMBL Nucleotide Sequence Database accession number LT009451.1 but has been adapted according to the sequence analysis results.
Other names of the plasmid are pLenti6(bla) -FLAG-dest, pLenti6Tag-blast and pLenti6-Flag-blast.
|EMBL Accession number:||LT009451.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||21/10/2019|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: ApaI/SpeI, HindIII, KpnI, PvuII and SacII.
The region from the ccdB gene up to the SV40 early promoter has been sequenced by BCCM/GeneCorner.
This plasmid has also been fully sequenced.
|History of deposit:||The plasmid was deposited by Dr P. De Groote(1)(2) and Prof. Dr W. Declercq(1)(2).
(1) Inflammation Research Center, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||De Groote et al., BioTechniques 60 (2016), 252-259 [PMID: 27177818] [DOI: 10.2144/000114417]
|Related plasmid reference:||PhD thesis Philippe De Groote (2012)
|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 (34 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.