pCasper3-GR vector

cat.# FP971

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 pCasper3-GR FP971 20 μg € 400 / 200* *50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer. The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type mammalian expression vector Reporter Casper3-GR Reporter codon usage mammalian Promoter for Casper3-GR PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use Expression of fluorescent caspase-3 apoptosis sensor Casper3-GR in mammalian cells under the control of CMV promoter; source of Casper3-GR coding sequence

Vector description

pCasper3-GR is a mammalian expression vector encoding a fluorescent sensor Casper3-GR (see reporter description). To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the Casper3-GR coding sequence [Kozak, 1987].

The vector can be also used as a source of Casper3-GR coding sequence. Flanking restriction sites are convenient for excision of Casper3-GR sequence and its further insertion into other expression vectors of choice. Alternatively, Casper3-GR 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

pCasper3-GR vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive Casper3-GR 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
Casper3-GR
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
TagRFP: 679-1374
Linker: 1375-1425
TagGFP: 1426-2130
Stop codon: 2131-2133
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2286-2291 & 2315-2320
mRNA 3' ends: 2324 & 2336
f1 single-strand DNA origin: 2383-2838
Bacterial promoter for expression of Kan^r gene
-35 region: 2900-2905
-10 region: 2923-2928
Transcription start point: 2935
SV40 origin of replication: 3179-3314
SV40 early promoter
Enhancer (72-bp tandem repeats): 3012-3083 & 3084-3155
21-bp repeats: 3159-3179, 3180-3200 & 3202-3222
Early promoter element: 3235-3241
Major transcription start points: 3231, 3269, 3275 & 3280
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3363-3365
Stop codon: 4155-4157
G->A mutation to remove Pst I site: 3545
C->A (Arg to Ser) mutation to remove BssH II site: 3891
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4393-4398 & 4406-4411
pUC plasmid replication origin: 4742-5385