PZY101 Map

Description:

This binary vector, also named PTF101, is used as a standard plant transformation vector for our transformation service. The vector was made previously that was derived from pPZP202 (Hajdukiewicz et al., 1994, Plant Mol Biol 25:989-994) and has been proven to work well for the transformation of soybean (Zeng et al., 2004, Plant Cell Rep 22:478-482; Paz et al., 2004, Euphytica 136:167-179) and maize (Frame et al., Plant Physiol 129:13-22). You can also use the sequence of this vector for designing your PCR or sequence primers.

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 35S promoter will allow you to insert your cassette. This 2X35S 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. Please let us know when you need this vector, and you can stop by to pick it up.

Special note:

1. Please 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. We have experienced this problem in several occasions. Therefore, we will appreciate your cooperation.
2. In order to use the EcoR I site within the MCS, a minimal ratio of this enzyme versus DNA is recommended to avoid excision of a "hidden" EcoR I site adjacent to the Xho I. When Hind III and EcoR I 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.