pCase12-mem vector

cat.# FP993

The vector sequence has been compiled using the information from sequence databases, published literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not been completely sequenced.

Download vector information: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price pCase12-mem FP993 20 μg € 600 The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. The product is not available in certain countries. Please contact Evrogen or your local distributor before ordering. Vector type mammalian expression vector Reporter Case12 Reporter codon usage mammalian Promoter for Case12 PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use Expression of membrane-targeted fluorescent Ca2+ sensor Case12 in mammalian cells under the control of CMV promoter; source of membrane-targeted Case12 coding sequence

Vector description

pCase12-mem is a mammalian expression vector encoding membrane-targeted fluorescent Ca2+ sensor Case12 (see reporter description). Case12 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Membrane localization signal (MLS) of neuromodulin is linked to the Case12 N-terminus. The MLS (N-terminal 20 amino acid residues of neuromodulin) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets Case12 to cellular membranes [Skene and Virag, 1989].

Membrane-localized Case12 expressed in 293T cell.

pCase12-mem vector can be used as a source of MLS-Case12 hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, MLS-Case12 coding sequence can be amplified by PCR.

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-mem 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.

Location of features

P_{CMV IE}: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Case12-mem fusion
Start codon (ATG): 679-681
Neuromodulin N-terminal sequence (mem): 679-738
Start of Case12 coding sequence (ATG): 739-741
Stop codon: 1978-1980
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1976-1981, 2134-2139 & 2163-2168
mRNA 3' ends: 2172 & 2184
f1 single-strand DNA origin: 2231-2686
Bacterial promoter for expression of Kan^r gene
-35 region: 2748-2753
-10 region: 2771-2776
Transcription start point: 2783
SV40 origin of replication: 3027-3162
SV40 early promoter
Enhancer (72-bp tandem repeats): 2860-2931 & 2932-3003
21-bp repeats: 3007-3027, 3028-3048 & 3050-3070
Early promoter element: 3083-3089
Major transcription start points: 3079, 3117, 3123 & 3128
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3211-3213
Stop codon: 4003-4005
G->A mutation to remove Pst I site: 3393
C->A (Arg to Ser) mutation to remove BssH II site: 3739
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4241-4246 & 4254-4259
pUC plasmid replication origin: 4590-5233