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GeneCorner plasmid details

Last data update: 21 October 2020 04:26 CEST

Plasmid name: pCAGGS-E-hTRAM (LMBP 5227)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p5227.gb (View with Genome Compiler)
p5227.txt
p5227.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human TRIF-related adapter molecule cDNA (TICAM2, hTRAM)
E-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-EmMyD88L
Further information: The plasmid was constructed as follows: the human TRIF-related adapter molecule coding sequence (hTRAM) was inserted as a NotI/XhoI PCR fragment between the NotI and XhoI sites of pCAGGS-EmMyD88L, in frame with the N-terminal E-tag.
The XbaI site located in the AG promoter sequence is removed (filled-in and religated).
pCAGGS-EhTRAM is useful for highly efficient expression of hTRAM 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726979.1.
The nucleotide sequence of the hTRAM cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AY232653.1.
Other names of the plasmid are pCAGGS-EhTRAM and pCAGGS-E-hTRAM(zXbaI).
EMBL Accession number: AY232653.1, view at EMBL, GenBank, DDBJ
LT726979.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 01/02/2007
Sequence detail:
Nucleotide sequence at the borders of the fusion gene:

                   ---------------------- E-tag ----------------------
5' ... TTCCACC.ATG.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAC.CCG.CTG.GAA.CCG.CGT.GCG. 
               Met Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala
               ***                                                     NotI
            NcoI
            

               -------------- hTRAM ----------------->            <-- pCAGGS
       GCC.GCA.ATG.GGT.ATC.GGG ... CAA.TTT.ATT.GCC.TGA.CTCGAGCTGCAGCCAATGCCCTGG ... 3'
       Ala Ala Met Gly Ile Gly     Gln Phe Ile Ala +++ 
               ***                                     XhoI  PstI                    


forward primer: 5' ATAAGAATGCGGCCGCA.ATG.GGT.ATC.GGG.AAG.TCT 3'
                           NotI

reverse primer: 5' CCGCTCGAG.TCA.GGC.AAT.AAA.TTG.TCT.TTG.TAC.C 3'
                      XhoI
***: start codon.
+++: termination codon.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BalI, BglII, EcoRI, HindII, PstI, NotI and XhoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr D. Vercammen(1) (2) and 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: Maelfait et al., J. Exp. Med. 205 (2008), 1967-1973 [PMID: 18725521] [DOI: 10.1084/jem.20071632]
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-E-hTRAM (LMBP 5227) is available at BCCM/GeneCorner. This plasmid was deposited by Dr D. Vercammen and Prof. Dr R. Beyaert and was published in Maelfait et al., 2008.

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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