pmKate2-endo vector

cat.# FP314

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 pmKate2-endo FP314 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 mKate2 Reporter codon usage mammalian Promoter for mKate2 PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use far-red fluorescent labeling of vesicles of the endocytic pathway

Vector description

pmKate2-endo is a mammalian expression vector intended for far-red fluorescent labeling of vesicles of the endocytic pathway [Adamson et al., 1992], allowing the monitoring of intracellular membrane traffic during endocytosis in living cells. The vector encodes far-red fluorescent protein mKate2 (see reporter description) targeted to endosomes by human RhoB GTPase fused to the mKate2 C-terminus. The fusion also contains c-Myc epitope tag.

HeLa cell expressing fusion of human RhoB GTPase and c-Myc epitope tag with mKate2. The fusion protein is localized in the endosomes.

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].

pmKate2-endo vector can be used as a source of mKate2-RhoB hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.

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

pmKate2-endo vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-RhoB fusion 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
mKate2-RhoB GTPase fusion
Start codon (ATG): 613-615
Start of mKate2 coding sequence (ATG): 613-615
Last amino acid in mKate2: 1312-1314
c-Myc epitope: 1357-1359
RhoB GTPase: 1383-1968
Stop codon: 1969-1971
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2163-2168 & 2192-2197
mRNA 3' ends: 2201 & 2213
f1 single-strand DNA origin: 2260-2715
Bacterial promoter for expression of Kan^r gene
-35 region: 2777-2782
-10 region: 2800-2805
Transcription start point: 2812
SV40 origin of replication: 3056-3191
SV40 early promoter
Enhancer (72-bp tandem repeats): 2889-2960 & 2961-3032
21-bp repeats: 3036-3056, 3057-3077 & 3079-3099
Early promoter element: 3112-3118
Major transcription start points: 3108, 3146, 3152 & 3157
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3240-3242
Stop codon: 4032-4034
G->A mutation to remove Pst I site: 3422
C->A (Arg to Ser) mutation to remove BssH II site: 3768
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4270-4275 & 4283-4288
pUC plasmid replication origin: 4619-5262