Last data update: 24 January 2024 16:39 CET
Plasmid name: pDS56HTNFm29S (LMBP 2650)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p2650.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human tumor necrosis factor cDNA (TNF); mutated mature sequence |
| Promoter: | Phage T5 N25 promoter with two lac operator sequences (N25/O2) |
| Ribosome binding site: |
Ribosome binding site RBSII; synthetic sequence |
| Terminator: | Escherichia coli rrnB operon T1 terminator |
| Selection marker: | Ampicillin (amp) Chloramphenicol (cam) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pDS56HTNF; pMcT4HTNFm29S |
| Further information: | The plasmid was constructed by inserting the AvaI-BstEII fragment from pMcT4HTNFm29S, containing the mutated mature hTNF sequence, between the AvaI and BstEII sites of pDS56HTNF. Codon 29 (CTG) of hTNF encoding leucine (L) is mutated to TCC encoding serine (S), resulting in the loss of an AlwNI site, by which AlwNI became unique. The unique NruI site of pDS56HTNF (due to silent mutations of AA17 and 18) is lost by the exchange of the fragment. Expression of hTNF is rapidly induced by addition of IPTG, which inactivates the lacI repressor. The promoter/operator element T5 N25/O2 consists of the highly efficient E. coli phage T5 N25 promoter and two lac operator sequences (Bujard et al. 1987). The presence of the lacI(q) repressor gene is necessary to control the activity of the promoter. The synthetic ribosome binding site RBSII is described in Stüber et al. (1990). The nucleotide sequence of the wt hTNF cDNA corresponds with the EMBL Nucleotide Sequence Database accession number X01394.1. Other name of the plasmid is pDS56/RBSII,SphI-TNFalpham29S. |
| EMBL Accession number: | X01394.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 23/09/1993 |
| Sequence detail: | pMcT4HTNF (hTNF wild type)
17 18 19 20 26 27 28 29 30 31 32 33 34 35 36 37 38 39
5' ... GTA.GCA.AAC.CCT ... CTC.CAG.TGG.CTG.AAC.CGC.CGG.GCC.AAT.GCC.CTC.CTG.GCC.AAT ... 3'
Val Ala Asn Pro Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn
AlwNI XcmI BglI BalI
EaeI
BstXI
pDS56HTNF
17 18 19 20 26 27 28 29 30 31 32 33 34 35 36 37 38 39
5' ... GTC.GCG.AAC.CCT ... CTC.CAG.TGG.CTG.AAC.CGC.CGG.GCC.AAT.GCC.CTC.CTG.GCC.AAT ... 3'
Val Ala Asn Pro Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn
^ ^ AlwNI XcmI BglI BalI
NruI EaeI
BstXI
pDS56HTNFm29S
17 18 19 20 26 27 28 29 30 31 32 33 34 35 36 37 38 39
5' ... GTA.GCA.AAC.CCT ... CTC.CAG.TGG.TCC.AAC.CGC.CGG.GCC.AAT.GCC.CTC.CTG.GCC.AAT ... 3'
Val Ala Asn Pro Leu Gln Trp Ser Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn
^^^ XcmI BglI BalI
EaeI
^: mutated nucleotide. BstXI
Punctuation indicates reading frame. |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr R. Beyaert(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Related plasmid reference: | Stüber et al., Eur. J. Immunol. 20 (1990), 819-824 [PMID: 1693336] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 M15[pREP4] |
| Host reference: | Beckwith et al., Mol. Biol. 8 (1964), 427-430 [PMID: 14168696] |
| Helper plasmid: | pREP4 |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | Escherichia coli SG13009 is also a suitable host. |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pDS56HTNFm29S (LMBP 2650) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert . |
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.