Template:JTK Arkin-TransposonDesignAndAssembly

This part is used in a project to assemble two different transposons on suicide plasmids.

Transposon/Plasmid 1
The first transposon/plasmid combination has a smaller transposable element which includes a spectinomicin resistance marker, a promoterless gentimicin marker (for selection of transposons that land inside a transcribed region), and a T7 promoter to help determine the region that the transposon inserted into. The rest of the plasmid contains a chloramphenicol resistance marker, a tranposase generating casette, an origin of transfer, and a conditional origin of replication (see box below about construction of transposon plasmids).
Preliminary experiments suggest that the suicide plasmid delivers the transposon with moderate efficiency, but that the ribosome binding site on the gentimicin gene is too weak to confer resistance from genomic transcription.


Transposon/Plasmid 2
The second transposon/plasmid combination has a larger transposable element which includes a chloramphenicol resistance marker, a promoterless gentimicin marker (for selection of transposons that land inside a transcribed region), and conditional origin of replication, and a T7 promoter. The FRT sites are an artifact of construction, and don't have any function in this case. Moving the origin of replication inside the transposon can greatly ease the process of decoding where the transposon inserted. The rest of the plasmid contains a spectinomycin resistance marker, a tranposase generating casette, and an origin of transfer.
Preliminary experiments suggest that the transposon is delivered with very poor efficiency, perhaps as a consequence of its significant size.