Last data update: 12 August 2020 04:29 CEST
Plasmid name: pAGRIKOLA-CATMAXyZZZZZ (LMBP 5554)
|Price category:||Cat. 2 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Arabidopsis thaliana gene-specific tag (GST)|
|Promoter:||Cauliflower mosaic virus 35S promoter (CaMV 35S)
Agrobacterium tumefaciens Ti-plasmid nopaline synthase promoter (nos)
Escherichia coli lac operon promoter
Phage SP6 promoter
Phage T7 gene 10 promoter (T7g10)
|Terminator:||Agrobacterium tumefaciens Ti-plasmid octopine synthase terminator (ocs)
Agrobacterium tumefaciens Ti-plasmid nopaline synthase terminator (nos)
|Selection marker:||Neomycin (neo; kanamycin (kan))
|Replicon:||Escherichia coli plasmid pMB1 origin
Escherichia coli plasmid pSa origin
|Host range:||Escherichia coli
|Further information:||This datasheet is representative for all the pAGRIKOLA-CATMA plasmids available at BCCM/GeneCorner under library accession number LMBP LIB27. Consequently, the accession number 'LMBP 5554' of this template clone is fictitious.
pAGRIKOLA-CATMAXyZZZZZ is a hairpin RNA Gateway expression vector containing authenticated A. thaliana GST's. The plasmid is used for gene silencing in plant cells.
In the framework of the European AGRIKOLA programme, the GST amplified from Arabidopsis genomic DNA was inserted in the pDONR207 vector (Invitrogen) using the Gateway BP reaction, resulting in an Entry clone. The Entry clone was then recombined with pAGRIKOLA,1 in a double LR clonase reaction. The cloning protocols are described on the AGRIKOLA web site (www.agrikola.org).
Because the double LR recombination could produce different intermediates depending on which repeats recombined with each other, the intron spaces in the expression clone either retained their original orientation or were flipped. As the two head-to-head introns are spliced out in plant cells, efficient silencing is guaranteed.
The plasmid contains the bar gene for basta herbicide resistance selection in transformed plant cells.
Information of the characterised GST is available in the online CATMA database (http://www.catma.org).
A list of the validated pAGRIKOLA-CATMA plasmids can be found on http://bccm.belspo.be/catalogues/genecorner-dna-libraries/genecorner-gst-clones.
In the compiled nucleotide sequence of this template plasmid, the GST sequences are each replaced by a stretch of 200 'N' nucleotides (CATMAXyZZZZZ), flanked by the attB1 and attB2 recombination sites. Note that in the GST amplicons, the Arabidopsis gDNA is bracketted by a 17-nucleotide adaptor on either side (for more information see http://www.catma.org/extensions.html).
The nucleotide sequence of pAGRIKOLA (parental clone of pAGRIKOLA,1) corresponds with the EMBL Nucleotide Sequence Database accession number AY568055.1.
|EMBL Accession number:||AY568055.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||16/02/2007|
Agri 51 primer: 5' CAA CCA CGT CTT CAA AGC AA 3' Agri 56 primer: 5' CTG GGG TAC CGA ATT CCT C 3' Agri 64 primer: 5' CTT GCG CTG CAG TTA TCA TC 3' Agri 69 primer: 5' AGG CGT CTC GCA TAT CTC AT 3'
|Authenticity test:||The criteria to authenticate the pAGRIKOLA hairpin RNA expression clones are:
(1) the presence of two attB1-GST-attB2 cassettes into the pAGRIKOLA-derived expression clone with a size compatible with the expected GST, as demonstrated by the DNA gel analysis of PCR amplicons;
(2) a GST sequence obtained from the purified hairpin expression clone that shares at least 95% homology with the expected GST, over an alignment of at least 60 nucleotides.
|History of deposit:||This plasmid was deposited by the Functional Genomics Division(1) on behalf of the AGRIKOLA consortium(2).
(1) Department of Plant Systems Biology, Ghent University, Belgium
|Plasmid reference:||Hilson et al., Genome Res. 14 (2004), 2176-2189 [PMID: 15489341]
|Related plasmid reference:||PhD thesis Heidrich et al. (2012)
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
|Cultivation medium:||LB-Lennox + kanamycin (50 μg/ml)|
|Other culture collection numbers:||-|
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.