pKillerOrange-dMito vector

cat.# FP224

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)

Product Cat.# Size Price pKillerOrange-dMito FP224 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 KillerOrange Reporter codon usage mammalian Promoter for KillerOrange PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use Expression of mitochondria-targeted KillerOrange in mammalian cells under the control of CMV promoter; source of mitochondria-targeted KillerOrange coding sequence

Vector description

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

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

pKillerOrange-dMito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted KillerOrange 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
KillerOrange-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 KillerOrange coding sequence(GAG): 801-803
Stop codon: 1509-1511
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1664-1669 & 1693-1698
mRNA 3' ends: 1702 & 1714
f1 single-strand DNA origin: 1761-2216
Bacterial promoter for expression of Kan^r gene
-35 region: 2278-2283
-10 region: 2301-2306
Transcription start point: 2313
SV40 origin of replication: 2557-2692
SV40 early promoter
Enhancer (72-bp tandem repeats): 2390-2461 & 2462-2533
21-bp repeats: 2537-2557, 2558-2578 & 2580-2600
Early promoter element: 2613-2619
Major transcription start points: 2609, 2647, 2653 & 2658
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2741-2743
Stop codon: 3533-3535
G->A mutation to remove Pst I site: 2923
C->A (Arg to Ser) mutation to remove BssH II site: 3269
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3771-3776 & 3784-3789
pUC plasmid replication origin: 4120-4763