Last data update: 24 January 2024 16:39 CET
Plasmid name: pCAGGS-Flag-hABIN1-P299A (LMBP 5132)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p5132.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1, NAF1); mutated sequence FLAG epitope tag; N-terminal |
| Promoter: | Chicken β-actin/rabbit β-globin hybrid promoter (AG) Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Rabbit β-globin polyadenylation signal (β-globin polyA) 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: | pCAGGS-E-hABIN1-P299A; pCAGGS-FLAGmA20 |
| Further information: | The plasmid was constructed by ligating the three following fragments: 1) the 2215 bp ScaI/KpnI fragment from pCAGGS-FLAGmA20 2) a 315 bp KpnI/SphI PCR fragment using pCAGGS-E-hABIN1-P299A as template 3) the 4225 bp SphI/ScaI fragment from pCAGGS-E-hABIN1-P299A. As compared to the wt human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) cDNA, the proline (P) codon at position 299 is replaced by an alanine (A) codon resulting in the loss of an AvaI site and the isoleucine (I) codon at position 9 contains a silent mutation (atc -> att) resulting in the loss of a DpnI and a Sau3AI site. pCAGGS-Flag-hABIN1-P299A is useful for highly efficient expression of hABIN-1m, fused in phase to the N-terminal FLAG epitope tag, under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells. The AG promoter sequence consists of the chicken β-actin promoter, the first exon and part of the first intron (that seems to have a strong enhancer-like activity) linked to a rabbit β-globin fragment, consisting of a 3' part of the second intron (inclusive a branch point which is required for normal splicing reactions) and a 5' part of the third exon. When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide. The nucleotide sequence of the hABIN-1 cDNA was obtained from Genbank (Accession number NM_006058.2). Other name of the plasmid is pCAGGS-Flag-hABIN1-FLX. |
| EMBL Accession number: | NM_006058.2, view at GenBank |
| Latest sequence update: | 03/01/2006 |
| Sequence detail: | Primers used to amplify the KpnI-SphI fragment of the hABIN-1 coding sequence:
1
forward: 5' ... GGGGTACC.ATG.GAA.GGG.AGA.GGA.CCG ... 3'
KpnI ***
NcoI
375
reverse: 5' ... GCCGCTCGAG.TCA.TTC.AAT.CTT.GGA.CTT.GGC ... 3'
XhoI +++
7 9 11 297 299 301
WT (NM_006058): 5' ... TAC.CGG.ATC.TAC.GAC ... GGC.GCA.CCC.GAG.AAG ... 3'
Tyr Arg Ile Tyr Asp Gly Ala Pro Glu Lys
DpnI AvaI
Sau3AI
7 9 11 297 299 301
P299A mutant: 5' ... TAC.CGG.ATT.TAC.GAC ... GGC.GCA.GCC.GAG.AAG ... 3'
Tyr Arg Ile Tyr Asp Gly Ala Ala Glu Lys
^ ^
***: start codon.
+++: termination codon.
^: mutated nucleotide.
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: | - |
| 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 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pCAGGS-Flag-hABIN1-P299A (LMBP 5132) 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.