Replicative minicircle vectors with improved expression
The present invention relates to the production and use of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules.
1. A eukaryotic replicative minicircle expression vector comprising: i) a eukaryotic region sequence with 5′ and 3′ ends encoding a gene of interest and comprising an intron with a 5′ splice donor and a 3′ acceptor branch site, and a 3′ UTR, ii) a spacer region of less than 500 basepairs in length linking the 5′ and 3′ ends of the eukaryotic region sequences, iii) a bacterial replication origin positioned at a first location selected from the group consisting of within said spacer region, within said 3′ UTR and within said intron between the 5′ splice donor and the 3′ acceptor branch site, and iv) a RNA selectable marker positioned at a second location selected from the group consisting of within said spacer region, within said 3′ UTR and within said intron between the 5′ splice donor and the 3′ acceptor branch site, wherein the first location is different than the second location, wherein the bacterial replication origin is an R6K bacterial replication origin, and wherein the R6K bacterial replication origin has at least 95% sequence identity to the sequence of SEQ ID NO: 11.
2. The vector of claim 1 , wherein said RNA selectable marker is a functional RNA-OUT with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 22.
3. The vector of claim 1 , wherein said RNA selectable marker is selected from the group consisting of an RNA-OUT selectable marker that encodes functional RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21, an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33, an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35 and a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA with at least 95% sequence identity to SEQ ID NO: 38.
4. The vector of claim 1 , wherein said intron with a 5′ splice donor and a 3′ acceptor branch site is a functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.