Genes of interest were cloned into the multiple cloning sites of pUT18 and pKT2538 using Gibson Assembly.
To generate the ins:BB1.0L; cryaa RFP construct, an insulin promoter containing vector with multiple cloning sites (MCS) (ins:MCS; cryaa RFP) was first generated.
Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites.
This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp.
The pOKD4 and pOKD5 vectors have been engineered to possess exactly the same multiple cloning sites as pET26b thus allowing for the relatively easy shuttling of genes to and fro.
All of the vectors have extensive multiple cloning sites, and versions are available for producing N- or C- terminal GFP fusions at each of the possible relative expression levels.
The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5′ integral intron for efficient gene expression.
Our new vector introduces the histidine tag at the donor level and places the multiple cloning sites within the attL recombination sites producing cleavable histidine-tagged proteins with a short, neutral linker of five residues.
Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI.
A cloning vector for lactic acid bacteria was developed on the basis of the pC7 replicon, into which were inserted an erythromycin resistance gene as a marker, multiple cloning sites, and Escherichia coli ColE1 replication origin.
RBS, ribosome-binding site; MCS, multiple cloning sites; ORF, open reading frame.
