Last data update: 24 January 2024 16:39 CET
Plasmid name: pSV23SmTNFrPDI (LMBP 3411)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3411.gb
(View with Genome Compiler) p3411.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Mouse tumor necrosis factor cDNA (Tnf, TNF) Rat protein disulfide isomerase cDNA (rPDI); fragment |
| Promoter: | Simian virus 40 early promoter (SV40 early) |
| Ribosome binding site: |
- |
| Terminator: | Simian virus 40 polyadenylation signal (SV40 polyA) |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin Simian virus 40 bidirectional origin (SV40) |
| Host range: | Escherichia coli Mammalian cells; SV40 permissive cells |
| Parental clone: | pSV23SMTNF; ppdi100; pMaMTNF |
| Further information: | This plasmid was constructed as follows: 1) the unique BamHI and XbaI sites of pSV23SMTNF were eliminated by filling-in with Klenow DNA polymerase and religation. 4) On the other hand, a 1000 bp EcoRI-BamHI fragment of ppdi100, containing the carboxy terminal part of the rPDI gene was inserted in pMa58 and an XbaI site was created by site-specific mutagenesis. 5) The XbaI-BamHI fragment of this intermediate construct, containing the last 14 codons of rat PDI and part of the 3' untranslated region, was inserted between the newly created XbaI and BamHI sites of the pSV23SmTNF mutant. 2) The termination codon of mTNF was removed and an XbaI and BamHI site were introduced by direct mutagensis of pMaMTNF. 3) This mutated mTNF gene was inserted as a SalI fragment between the SalI sites of the modified pSV23SMTNF, replacing the wild type mTNF sequence. 6) A BglII-BamHI digestion eliminated the 3' untranslated region part of the rat PDI gene. The extention of the mTNF gene with the last 14 codons of the rat PDI gene might result in the retention of TNF in the endoplasmic reticulum. The plasmid contains the SV40 small t-antigen splicing signal. Restriction enzyme digestions revealed that approx. 60 nucleotides are missing from the 3' untranslated region of the mTNF gene, including the 3'-terminal (supposedly restored) EcoRI site. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727484.1. Other name of the plasmid is pSV23SmTNF.PDI. |
| EMBL Accession number: | M11731, view at EMBL, GenBank, DDBJ LT727484.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 04/03/1996 |
| Sequence detail: | Introduction of an XbaI and BamHI site at the termination codon of mTNF:
157
mTNFwt :5' GTC.ATT.GCT.CTG.TGAAGGGAATGGGTGTTC 3'
mutated mTNF :5' GTC.ATT.GCT.CTG.TCT.AGA.GGA.TCC.GTG.TTC 3'
XbaI BamHI
Introduction of an XbaI site at amino acid codon 473 of rPDI :
474 475 489
5' GAG.TCT.AGA.ATG.GGA.GAA..........CTG.TAG.TGCAGAAGCCAGATCTGGGC 3'
XbaI BglII
Fusion region between murine TNF and rat PDI :
mTNF -->| | rat PDI | 3 UTR mTNF
157 475 489
5' ATT.GCT.CTG.TCT.AGA.ATG.GAA......CTG.TAG.TGCAGAAGCCAGATCCGTGT.. 3'
------
BglII/BamHI
fusion |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BamHI/XbaI, BglII/NcoI, EcoRI/PstI, HaeII, SalI, TaqI and XmnI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr E. Decoster(1)(2) and Prof. Dr W. Fiers(1)(2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 MC1061 |
| Host reference: | Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Related host reference: | Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSV23SmTNFrPDI (LMBP 3411) is available at BCCM/GeneCorner. This plasmid was deposited by Dr E. Decoster and Prof. Dr W. Fiers. |
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.