Last data update: 24 January 2024 16:39 CET
Plasmid name: pDG2i-hRIP4-K51R-FLAG-puro (LMBP 7948)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p7948.gb
(View with Genome Compiler) p7948.txt p7948.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human receptor-interacting serine-threonine kinase 4 cDNA (RIPK4, RIP4, ANKRD3, ANKK2, DIK, PKK, GeneID 54101); mutated sequence FLAG epitope tag; C-terminal Reverse tetracycline-responsive transcriptional activator (rtTA); variant 3 (rtTA3) |
| 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 |
| 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) |
| Replicon: | Simian virus 40 bidirectional origin (SV40) Escherichia coli plasmid pMB1 origin Phage f1 origin |
| Host range: | Escherichia coli Mammalian cells; SV40 permissive cells |
| Parental clone: | pDG2i-Flag puro |
| Further information: | The plasmid was constructed by cloning the mutated human RIPK4 coding sequence into pDG2i-Flag puro via Gateway LR recombination. This is a lentiviral Gateway expression vector, with the tet-inducible minimal human CMV-IE promotor and rtTA3 on the same plasmid. The plasmid contains the human RIPK4 coding sequence with a K51R mutation. 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. Plasmid integrity should be checked via PvuII restriction digest after each subcultivation. The nucleotide sequence of the wild-type human RIPK4 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number BC110618.1. The nucleotide sequence of the parental pDG2i-Flag puro vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009444.1, except for the changes due to the NGS sequence analysis. Other name of the plasmid is pDG2hRIP4K51R_Flag puro. |
| EMBL Accession number: | LT009444.1, view at EMBL, GenBank, DDBJ BC110618.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 02/03/2020 |
| Sequence detail: | 45 50 51 55
hRIPK4 K51R mutant: ... AAG.ACC.TGG.CTC.GCG.ATC.AGG.TGC.TCG.CCC.AGC ...
Lys Thr Trp Leu Ala Ile Arg Cys Ser Pro Ser
^ ^ ^
NruI
45 50 51 55
hRIPK4 wild-type: ... AAG.ACC.TGG.CTG.GCC.ATC.AAG.TGC.TCG.CCC.AGC ...
Lys Thr Trp Leu Ala Ile Lys Cys Ser Pro Ser
Punctuation indicates reading frame.
^: mutated nucleotide. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: HindIII, NdeI, PvuII and SpeI/XhoI. |
| 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: | - |
| Related plasmid reference: | PhD thesis Philippe De Groote (2012) 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 K12 DH5α |
| Host reference: | Focus 8 (1986), 9 |
| Related host reference: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 28°C |
| Biosafety level: | L1 in E. coli; L2 in mammalian cells |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pDG2i-hRIP4-K51R-FLAG-puro (LMBP 7948) is available at BCCM/GeneCorner. This plasmid was deposited by Dr P. De Groote and Prof. Dr W. Declercq . |
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.