pKillerRed-dMito vector

cat.# FP964

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 pKillerRed-dMito FP964 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 KillerRed Reporter codon usage mammalian Promoter for KillerRed PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use Expression of mitochondria-targeted KillerRed in mammalian cells under the control of CMV promoter; source of mitochondria-targeted KillerRed coding sequence

Vector description

pKillerRed-dMito is a mammalian expression vector encoding mitochondria-targeted KillerRed (see reporter description). KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Duplicated mitochondrial targeting sequence (MTS) is fused to the KillerRed N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995].

pKillerRed-dMito vector can be used as a source of dMTS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, dMTS-KillerRed 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

pKillerRed-dMito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted KillerRed 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
KillerRed-dMito fusion
Start codon (ATG): 597-599
Mitochondrial localization signal 1 (MLS-1): 597-689
Mitochondrial localization signal 2 (MLS-2): 690-782
Start of KillerRed coding sequence (ATG): 798-800
Stop codon: 1515-1517
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1671-1676 & 1700-1705
mRNA 3' ends: 1709 & 1721
f1 single-strand DNA origin: 1768-2223
Bacterial promoter for expression of Kan^r gene
-35 region: 2285-2290
-10 region: 2308-2313
Transcription start point: 2320
SV40 origin of replication: 2564-2699
SV40 early promoter
Enhancer (72-bp tandem repeats): 2397-2468 & 2469-2540
21-bp repeats: 2544-2564, 2565-2585 & 2587-2607
Early promoter element: 2620-2626
Major transcription start points: 2616, 2654, 2660 & 2665
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2748-2750
Stop codon: 3540-3542
G->A mutation to remove Pst I site: 2930
C->A (Arg to Ser) mutation to remove BssH II site: 3276
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3778-3783 & 3791-3796
pUC plasmid replication origin: 4127-4770