Rolling circle replicon expression vector

Abstract

A rolling circle DNA replicon which replicates in a host eukaryotic cell is disclosed which has a truncated replication cycle. The rolling circle DNA replicon comprises the following elements present on the same DNA molecule. It contains a Rep gene open reading frame from a virus belonging to the viral taxonomic families Geminiviridae, Circoviridae or genus Nanovirus. The Rep gene open reading frame is placed under transcriptional control of a promoter, which is placed 5′ of the gene. Any sequences that are required to be present in cis on the rolling circle DNA replicon in order that the Rep protein might promote replication of the rolling circle DNA replicon are included. An expression cassette for expression of an ancillary protein that is capable of creating a cellular environment permissive for replication of the rolling circle DNA replicon in the host cell of interest is also included. At least one expression cassette with an RNA polymerase II promoter, a multiple cloning site, and transcription termination and polyadenylation signals suitable for transcription of RNA molecules not normally intrinsic to a geminiviral, circoviral or nanoviral genome is also included.

1 . A rolling circle DNA replicon which replicates in a host eukaryotic cell which replicon has a truncated replication cycle, said rolling circle DNA replicon, comprising the following elements, present on the same DNA molecule: 
 a) a Rep gene open reading frame from a virus belonging to the viral taxonomic families Geminiviridae, Circoviridae or genus  Nanovirus,  said Rep gene open reading frame is placed under transcriptional control of a promoter, which promoter is placed 5′ of the gene;    b) sequences that are required to be present in cis on the rolling circle DNA replicon in order that a Rep protein might promote replication of the rolling circle DNA replicon;    c) an expression cassette for expression of an ancillary protein that is capable of creating a cellular environment permissive for replication of the rolling circle DNA replicon in the host cell of interest; and    d) at least one expression cassette with an RNA polymerase II promoter, a multiple cloning site, and transcription termination and polyadenylation signals suitable for transcription of RNA molecules not normally intrinsic to a Geminiviral, Circoviral or Nanoviral genome.    
     
     
         2 - 15 . (canceled)