Last data update: 24 January 2024 16:39 CET
Plasmid name: pcDNA1-hFADD-DED (LMBP 4606)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4606.gb
(View with Genome Compiler) p4606.txt p4606.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human Fas receptor-associated death domain cDNA (FADD); part containing the death effector domain (DED) E-tag; C-terminal |
| Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer Phage T7 gene 10 promoter (T7g10) Phage SP6 promoter |
| Ribosome binding site: |
- |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Suppressor tRNA gene (supF; requires a p3 containing host) |
| Replicon: | Bacteriophage M13 origin Escherichia coli plasmid pMB1 origin Polyoma virus origin Simian virus 40 bidirectional origin (SV40) |
| Host range: | Escherichia coli; use strains with the p3 helper plasmid (kan resistant, amber codon in amp and tet) Mammalian cells; SV40 permissive cells |
| Parental clone: | pcDNA1 |
| Further information: | The plasmid was constructed by inserting part of the hFADD coding sequence containing the death effector domain, picked up by reverse transcription PCR and provided with a C-terminal E-tag, between the XbaI and HindIII sites of pcDNA1. The hFADD-DED insert encodes amino acid 1 to 111, containing the death effector domain defined from codon 27 to 60. Sequence analysis revealed the absence of codon 86 to 89, without affecting the functionality of FADD-DED. pcDNA1-hFADD-DED is a shuttle vector designed for stable or high-level transient expression of human FADD-DED in mammalian cells. The inserted gene is expressed under control of the human CMV promoter and the SV40 polyadenylation site. The eukaryotic Kozak recognition sequence (CCGCC ATG G) for initiation of translation is present. The N-terminal DED-domain of hFADD allows the recruitment of caspase-8, and thereby the activation of the cysteine protease cascade. A small HIV (human immunodeficiency virus) fragment (29 nucleotides) between the CMV and T7 promoter sequences has an enhancer function. The T7 and SP6 promoters can be used for in vitro transcription of the cDNA insert. The presence of the polyoma and SV40 origins of replication enables the vector to replicate episomally in mammalian cells expressing large T-antigen (e.g. WOB, COS cells). The supF tRNA suppressor gene is used for selection in E. coli MC1061[p3]. It is recommended to select on both ampicillin and tetracycline containing media. The M13 origin is useful for synthesis of single-stranded DNA upon infection with the M13 helper phage. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727122.1. The nucleotide sequence of the hFADD cDNA corresponds with the EMBL Nucleotide Sequence Database accession number U24231.1. Other name of the plasmid is pCDNA1-hDED/FADD. |
| EMBL Accession number: | U24231.1, view at EMBL, GenBank, DDBJ LT727122.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 24/03/2009 |
| Sequence detail: | Nucleotide sequence of the fusion gene:
----------------- hFADD fragment -------------------------------->
---Kozak--- --- DED domain ---> |
1 2 26 27 28 59 60 61 84 85 90 91 92
5' ... CCCAAGCTTCCGCC.ATG.GAC ... CTA.TGC.CTC ... CAC.ACC.GAG ... GCG.GGG.GCC.GCG.CCT...
HindIII Met Asp Leu Cys Leu His Thr Glu Ala Gly Ala Ala Pro
***
NcoI
---------->
110 111 ---------------------- E-tag --------------------->
... AAA.GAT.GCG.GCC.GCA.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAT.CCG.CTG.GAA.CCG.CGT.GCC.GCA.TAG ... 3'
Lys Asp Ala Ala Ala Gly Ala Arg Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala +++
NotI
***: start codon.
+++: termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: NcoI, NdeI and NotI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof P. Vandenabeele(1) (2). It was constructed by G. Van Loo(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Boone et al., J. Biol. Chem. 275 (2000), 37596-37603 [PMID: 10988295] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 MC1061[p3] |
| Host reference: | - |
| Related host reference: | Instruction Manual (Invitrogen) Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Helper plasmid: | p3 |
| Cultivation medium: | LB-Lennox + ampicillin (50 μg/ml) + kanamycin (50 μg/ml) + tetracycline (10 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pcDNA1-hFADD-DED (LMBP 4606) is available at BCCM/GeneCorner. This plasmid was deposited by Prof P. Vandenabeele and was published in Boone et al., 2000. |
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.