Figure 1.

Proof-of-concept to establish successful transfer of large DNA fragments containing interspersed regions of AT-rich regulatory elements to a linear vector framework. (A). The schematic shows 7.5 kb and 9.5 kb fragments to be released from extant circular vectors pSN372 and pMG1847, respectively, for assembly into linear vectors. These fragments contain TetR- or TetR-DOZI-based translation regulation modules and a transcriptional unit in which expression of a FLuc reporter CDS is translationally controlled by TetR aptamers (shown as lollipops) located in either the 5′-UTR only or both 5′- and 3′-UTRs. (B) Strategy used to transfer the respective pSN372- and pMG1847-derived fragments into linear plasmids. The original pJAZZ-OC vector (Lucigen) was modified with a multi-cloning site gene block to create pSwing. To facilitate Gibson assembly, pSwing can be digested with restriction enzymes to expose regions homologous to cut pSN372- and pMG1847-derived fragments (red and green). (C) Restriction digestion analysis confirming proper topological assembly of pSwing, pSN372L and pSN1847L. For pSN1847L, several plasmids that do not contain the expected insert, and likely corresponding to pSwing, are indicated in red font.