pCase12-cyto is a mammalian expression vector encoding a fluorescent sensor Case12 (see reporter description). To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the Case12 coding sequence [Kozak, 1987].

The vector can be also used as a source of Case12 coding sequence. Flanking restriction sites are convenient for excision of Case12 sequence and its further insertion into other expression vectors of choice. Alternatively, Case12 coding sequence can be amplified by PCR.

Note: The plasmid DNA was isolated from dam⁺-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam^- host and make fresh DNA.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV IE}) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

SV40 early promoter (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

pCase12-cyto vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive Case12 expression in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].

Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.