To accommodate diverse tagging needs (different tag types and/or different locations), OriGene devised the novel PrecisionShuttleTM system to allow easy subcloning of an ORF from one tagged vector to another. The TrueORF Entry Vector contains C-terminal tags of Myc and DDK Tag* and is the entry vector for the PrecisionShuttle system. A large panel of destination vectors is available so that you can express the ORF with different tags, with tags at different ends of the protein, or as the native, untagged protein.
The key in the PrecisionShuttle system is the utilization of two rare-cutting restriction endonucleases, Sgf I and Mlu I. The entry vector and all destination vectors have identical multiple cloning sites (MCS) so that they can exchange the inserts through simple restriction digestion and ligation. In the TrueORF Entry Vector, the ORF is flanked by Sgf I (5’-end) and Mlu I (3’-end) sites. Digestion with these two enzymes releases the untagged ORF, which can then be ligated into a destination vector digested with the same enzymes. As all destination vectors carry the ampicillin resistance marker instead of the kanamycin resistance marker, the successfully shuttled product can be easily selected with ampicillin.
Schematic of the Precision Shuttling system.
Over 96% of human ORFs do not have internal Sgf I or Mlu I sites and are suitable for the above strategy. For the 4% of human ORFs that contains one or both enzyme sites, two additional restriction sites in the MCS (Asc I and Rsc II) can be used to shuttle the ORF into a destination vector. When this alternate strategy is required, that information is indicated in the description of the TrueORF clone.
Compared to the commonly used Gateway system, PrecisionShuttle has several major advantages:
- The entry vector can be used for tagged expression in mammalian cells and in cell-free systems. For many applications, there is no need to shuttle the ORF to a destination vector.
- The shuttling is accomplished with a simple restriction enzyme digestion/ligation without the need for an expensive recombination specific enzyme.
- There are no intellectual property restrictions for any users.
- Large ORFs up to 18Kb can be readily transferred using the PrecisionShuttle system while ORFs larger than 4Kb are unstable in recombination-based systems.
- The PrecisionShuttle vectors precisely add the intended tag to its desired location. There is no appendage of multiple amino acids on both ends of the protein.
* Peptide sequence of the DDK-tag (Flag®): N-DYKDDDDK-C
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