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GeneCorner plasmid details

Last data update: 22 October 2020 04:25 CEST

Plasmid name: pCAGGS-Flag-hABIN1-P299A (LMBP 5132)

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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.

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