Compositions of polynucleic acid vectors and uses thereof

A polynucleic acid destination vector comprising a backbone component and an insertion site component, wherein:

• (a) the backbone component comprises a nucleic acid sequence of a selectable marker, an origin of replication, and at least one Type IIS restriction site comprising a common recognition site and corresponding cleavage site, wherein the common recognition site is overlapped by a methylation site;
• and
• (b) the insertion site component comprises a 5′ Type IIS dual restriction site and a 3′ Type IIS dual restriction site, optionally, wherein the 5′ and 3′ Type IIS dual restriction sites are separated by at least one nucleotide;
• wherein each Type IIS dual restriction site comprises:
• (i) a first common recognition site and corresponding cleavage site, wherein the first common recognition site is overlapped by a methylation site that forms the border between the insertion site component and the backbone component and (ii) a second common recognition site and corresponding cleavage site, wherein the second recognition site lacks an overlapping methylation site, wherein the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site are both positioned between the first common recognition site and the second common recognition site, wherein:
• methylation of the destination vector at common recognition sites overlapped by a methylation site, blocks cleavage of the cleavage site corresponding to that common recognition site;
• exposure of the destination vector, when methylated, to a Type IIS restriction enzyme that recognizes the common recognition sites of the destination vector, generates two polynucleic acid fragments, wherein the terminal 5′ or 3′ nucleic acid overhangs of the fragment comprising the backbone component differ in nucleotide sequence;
• and
• the Type IIS cleavage sites in (a) differ from the Type IIS cleavage sites in (b) in nucleotide sequence.