DNA Plasmids with Improved Expression

Abstract

The present invention relates to the production and use of covalently closed circular (ccc) recombinant plasmids, and more particulary to vector modifications that improve express of said DNA molecules in the target organism.

Claimed is: 
     
         1 . An engineered bacterial cell comprising a Rep protein-dependent plasmid vector, a Rep protein gene that expresses a Rep protein, and a P L  promoter that controls the expression of the Rep protein gene, and wherein the P L  promoter comprises an OL1 mutation, wherein the OL1 mutation comprises either a single base substitution or a single base deletion that decreases or prevents repressor biding to OL1, and wherein the Rep protein-dependent plasmid vector comprises an R6K replication origin. 
     
     
         2 . The engineered bacterial cell of  claim 1 , wherein the P L  promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO: 44. 
     
     
         3 . The engineered bacterial cell of  claim 1 , wherein said Rep protein comprises an amino acid sequence of SEQ ID NO: 13, wherein the amino acid sequence comprises two mutations at amino acids 106 and 107 of SEQ ID NO:13. 
     
     
         4 . The engineered bacterial cell of  claim 1 , wherein the sequences of the P L  promoter comprising said OL1 mutation is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         5 . The engineered bacterial cell of  claim 1 , wherein the R6K replication origin comprises a nucleic acid sequence that possesses at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 22. 
     
     
         6 . The engineered bacterial cell of  claim 1 , wherein the Rep protein-dependent plasmid vector further comprises an RNA-OUT selectable marker. 
     
     
         7 . The engineered bacterial cell of  claim 6 , wherein the RNA-OUT selectable marker comprises a nucleic acid sequence with at least 90% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 36.