A Meganuclease-based transformation vector for use with long-DNA constructs or to assemble multiple genes into one construct.
Last modified October 8, 2004; information still current October 2004
What are meganucleases?
Meganucleses, also known as intron- or intein-encoded endonucleases, have long recognition sites, ranging from 9 to 19 base pairs long. Hence, they are extremely rare cutters.
What is pUGA?
pUGA is a transformation vector, whose multiple cloning site contains 5 meganuclease sites. These permit up to 9 different DNA constructs to be easily assembled into the vector.
How are multiple gene cassettes placed into pUGA?
pUGA is a “docking vector” with an associated series of shuttle vectors. These shuttle vectors have a multiple cloning site flanked by meganuclease sites. There are two types of shuttles:
- Heteroshuttles– have different meganuclease sites flanking the MCS. Cassette(s) of interest are first cloned into the shuttle. Meganucleases are then used to cut the cassettes out, and the DNA fragment can be inserted into pUGA without destroying the uniqueness of the meganuclease site within the MCS. Because most of the meganucleases sites are polar, the orientation of the cassette can be controlled Up to 4 heteroshuttles can dock into pUGA.
- Homoshuttles– have the same meganuclease sites flanking the MCS. Hence, up to 5 homoshuttles can dock into pUGA. However, the use of the homoshuttles destroys the uniqueness of the meganuclease site within pUGA.
FAQs:
What is the reference for pUGA?
- Thomson, J.M., P.R. LaFayette, M.A. Schmidt, and W.A. Parrott. 2002. Artificial gene-clusters engineered into plants using a vector system based on intron- and intein-encoded endonucleases. In Vitro-Plant 38:537-542. PDF.
- I-PpoI is available from Promega I-CeuI, I-PspI, and PI-SceI are available from NEBPI-TliI may be availabe by request from NEB
- NEB recommends that digests should be incubated in 0.5% SDS prior to electrophoresis
- Promega does not recommend any special treatment
- Are there any restrictions on the use of pUGA?pUGA and its shuttle plasmids are made available on a non-exclusive basis for non-commercial research uses, with the following conditions:
- These plasmids will not be transferred to any third parties without permission from their originators Any publications resulting from the use of these plasmids will acknowledge their use and cite the appropriate reference No guarantees, explicit or implied, on the effectiveness or efficiency of these plasmids, are made by their originators or The University of Georgia, nor can their originators or The University of Georgia be held liable for failure of the plasmids to perform as expected, or any consequences resulting therefrom
- Anyone desiring to use these plasmids for any commercial purpose must contact Shelley Fincher at The University of Georgia Technology Transfer Office for additional information.