Cloning vectors and vector components

Abstract

The present invention relates to systems, methods, and compositions for cloning and sequencing insert nucleic acid sequences. In particular, the present invention provides vectors and vector components configured for multiplex cloning, multiplex sequencing, and fixed orientation cloning. The present invention also provides vectors and vector components that allow insert sequences that are deleterious to a host cell to be successfully cloned.

1 . A composition comprising X+1 vector components, wherein each of said X+1 vector components are configured for combining in the presence of X+1 insert sequences to form a circular recombinant vector such that said X+1 vector components are non-contiguous within said circular recombinant vector.  
     
     
         2 . The composition of  claim 1 , wherein each of said X+1 vector components comprises; i) first and second free ends, and ii) a selectable marker region comprising at least one selectable marker sequence unique among said X+1 vector components.  
     
     
         3 . The composition of  claim 2 , wherein each of said X+1 vector components further comprises; iii) a first transcriptional terminator between said first free end and said selectable marker region, and iv) a second transcriptional terminator between said second free end and said selectable marker region.  
     
     
         4 . The composition of  claim 3 , wherein said first transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said first free end.  
     
     
         5 . The composition of  claim 3 , wherein said second transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said second free end.  
     
     
         6 . The composition of  claim 2 , wherein said selectable marker region in each of said X+1 vector components comprises a transcriptional terminator configured to terminate RNA transcripts encoded by at least one selectable marker sequence in said selectable marker region.  
     
     
         7 . The composition of  claim 2 , wherein each of said X+1 vector components comprises a first non-promoter sequence between said first free end and said selectable marker region, and a second non-promoter sequence between said second free end and said selectable marker region, wherein said first and second non-promoter sequences are unable to serve as an operable promoters in a host cell.  
     
     
         8 . The composition of  claim 2 , wherein at least one of said X+1 vector components comprises a promoter sequence between at least one of said first or second free ends and said selectable marker region, wherein said promoter sequence is capable of serving as an operable promoter in a host cell.  
     
     
         9 . The composition of  claim 2 , wherein said first and second free ends are non-compatible free ends.  
     
     
         10 . The composition of  claim 1 , wherein each of said X+1 vector components comprises two primer binding sites.  
     
     
         11 . The composition of  claim 1 , wherein each of said X+1 insert sequences comprise two identical sticky free ends that are unique among said X+1 insert sequences, wherein each of said X+1 vector components comprises two different sticky free ends, and wherein each of said two different sticky free ends binds one of said X+1 insert sequences.  
     
     
         12 - 28 . (cancelled).