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URL: http://www.addgene.org/73932

Proper Citation: RRID:Addgene_73932

Insert Name: bar

Organism: Synthetic

Bacterial Resistance: Spectinomycin

Defining Citation: PMID:15034747

Vector Backbone Description: Backbone Size:7132; Vector Backbone:pPZP201; Vector Types:Plant Expression; Bacterial Resistance:Spectinomycin

Comments: This vector is suitable hpRNA-mediated gene silencing in tissue (seed) specific manner. Please see the Plant Transformation Core Facility website for more information on using this plasmid: http://plantsci.missouri.edu/muptcf/pzy101.html The vector has following features: 1. It has two replicate origins that allow to replicate in both E. coli and Agrobacterium tumefaciens cells (at higher copy numbers). 2. It also has bom site that allows you to use tri-parental mating to mobilize the vector to Agrobacterium cells. 3. The aadA locus confers Spectinomycin (100 mg/L) or streptomycin (100 mg/L) resistance and thus enable to use either or both of these two antibiotics for the vector selection. 4. The multiple cloning site (MCS) adjacent to the double CaMV35S promoter will allow you to insert your cassette. This CaMV35S promoter is used to drive the bar ORF and, therefore, is not used in front to drive your gene of interest. You need to assemble your own expression cassette including promoter, gene coding sequence, and terminator, and then insert to anywhere between Hind III and EcoR I within the MCS. Special notes: 1. Insert your gene expression cassette in such a way that your promoter is adjacent to the promoter driving the bar gene selectable marker. This is to prevent transcription read-through from your gene expression cassette. 2. In order to use the EcoR I site within the MCS, a minimal ratio of this enzyme versus DNA is recommended to avoid star activity. When HindIII and EcoRI are in combined use, even less of each enzyme is required, and reaction should be conducted in a large volume to avoid excessive enzymatic activity.

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