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GeneCorner plasmid details

Last data update: 18 October 2021 09:15 CEST

Plasmid name: pDG2i-Flag puro (LMBP 7944)

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Price category: Cat. 3 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p7944.gb (View with Genome Compiler)
p7944.txt
p7944.pdf
Sequence
analysis results
Genecorner:

Sanger: 127470.fasta

Sanger: 127471.fasta

NGS: d01-gc-dec2018.fasta

Cloned DNA: B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)
Reverse tetracycline-responsive transcriptional activator (rtTA); variant 3 (rtTA3)
FLAG epitope tag; C-terminal
Promoter: Human cytomegalovirus immediate early promoter (CMV-IE); minimal promoter with tet responsive element (CMVTRE)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Simian virus 40 early promoter (SV40 early)
Escherichia coli lac operon promoter
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
Ribosome
binding site:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Simian virus 40 polyadenylation signal (SV40 polyA)
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)
Selection marker: Ampicillin (amp)
Puromycin (puro)
Chloramphenicol (cam)
Replicon: Simian virus 40 bidirectional origin (SV40)
Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli; use a ccdB-resistant strain for propagation
Mammalian cells; SV40 permissive cells
Parental clone: pLenti6-Flag puro; pTRIPZ
Further information: The construction of this plasmid is described in De Groote et al. (2016).
This is a lentiviral Gateway destination vector, with the tet-inducible minimal human CMV-IE promotor and rtTA3 on the same plasmid.
The plasmid is 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.
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).
Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA. Plasmid integrity should be checked via PvuII restriction digest after each subcultivation.
The nucleotide sequence of the pDG2i-Flag puro vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009444.1.
Other names of the plasmid are pDG2lenti_Flag rtTA3 puro, pDG2iTag-puro and pDG2i-Flag-puro.
EMBL Accession number: LT009444.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 05/01/2021
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: HindIII, NdeI, PvuII and SpeI/XhoI.

This plasmid has also been fully sequenced.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This 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: Afonina et al., EMBO Rep. 17 (2016), 914-927 [PMID: 27113748] [DOI: 10.15252/embr.201642109]
PhD thesis Philippe De Groote (2012)
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 (34 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: -

Refer in your Materials and Methods:

pDG2i-Flag puro (LMBP 7944) is available at BCCM/GeneCorner. This plasmid was deposited by Dr P. De Groote and Prof. Dr W. Declercq and was published in De Groote et al., 2016.

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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