During the cloning step of a PCR product in the StabyCloning^TM vector, the gene of interest is oriented and linked to the ccdA antidote gene (see the StabyCloning section). Using restriction sites in the pSTC1 vector, it is easy to transfer the gene of interest (linked to the ccdA antidote gene) into a sub-cloning vector. The ccdA presence linked to your gene allows selection of recombinants in CYS21 or SE1 bacteria (these bacteria contain a natural bacterial poison gene (encoding the poison protein CcdB counteracted by CcdA) into their chromosomes). Since this selection is very efficient, it is not required to purify your insert linked to ccdA gene before sub-cloning. The restriction-sites choice allows selection of insert orientation. After sub-cloning, the vector is ready for protein expression in SE1 bacteria.

The restriction sites were chosen to be compatible with sites of pSTC1 vector and to place your gene in frame with the protein tags. Several sub-cloning vectors were developed for each application: protein expression with his-tag, no tag, cherry tag, missing t-RNAs (see StabyCodon), yeast protein expression...

The multiple cloning sites of destination vectors contain the same compatible restriction sites. Consequently, one restriction of the pSCT1 vector is compatible with all StabySubCloning vectors.

Vector maps

The pSTC1.3 vector contains several restriction sites for sub-cloning steps. If ATG (start codon) of your gene are the first bases of your PCR insert in pSTC1.3, PmeI restriction site will place your gene in frame with the protein tags in the sub-cloning vectors.

StabyExpress sub-cloning vectors

-pSSC-Native1
Features:T7 promoter, no tag, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.

-pSSC-His1
Features: T7 promoter, His tag, Enterokinase cleavage site, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.

-pSSC-Cherry1
Features: T7 promoter, Cherry tag, Enterokinase cleavage site, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.

-pSSC-Native2
Features: T7 promoter, no tag, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.

-pSSC-His2
Features: T7 promoter, His tag, Enterokinase cleavage site, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.

-pSSC-Cherry2
Features: T7 promoter, Cherry tag, Enterokinase cleavage site, additional t-RNAs, Kan resistance, lacI repressor to reduce basal expression, plasmid stabilization in CYS21 and SE1 bacteria after sub-cloning.