pFusionRed-endo vector

cat.# FP427

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 pFusionRed-endo FP427 20 μg € 480 / 240* *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 FusionRed Reporter codon usage mammalian Promoter for FusionRed PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use red fluorescent labeling of vesicles of the endocytic pathway

Vector description

pFusionRed-endo is a mammalian expression vector intended for 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 red fluorescent protein FusionRed (see reporter description) targeted to endosomes by human RhoB GTPase fused to the FusionRed C-terminus. The fusion also contains c-Myc epitope tag.

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(A) – Human endosomes in normal African green monkey kidney fibroblast cells expressing . The compartments are seen shuttling back and forth on their membrane transport pathway between the plasma membrane and lysosomes. Movie from Shemiakina et al., 2012. (B) – HeLa cell expressing fusion of human RhoB GTPase and c-Myc epitope tag with FusionRed. The fusion protein is localized in the endosomes. Scale bar represents 10 μm. Data from Shemiakina et al., 2012

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

pFusionRed-endo vector can be used as a source of FusionRed-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

pFusionRed-endo vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-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
FusionRed-Human RhoB GTPase fusion
Start codon (ATG): 613-615
Start of FusionRed coding sequence (ATG): 613-615
Last amino acid in FusionRed: 1306-1308
c-Myc epitope: 1363-1395
Human RhoB GTPase: 1399-1977
Stop codon: 1975-1977
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2169-2174 & 2198-2203
mRNA 3' ends: 2207 & 2219
f1 single-strand DNA origin: 2266-2721
Bacterial promoter for expression of Kan^r gene
-35 region: 2783-2788
-10 region: 2806-2811
Transcription start point: 2818
SV40 origin of replication: 3062-3197
SV40 early promoter
Enhancer (72-bp tandem repeats): 2895-2966 & 2967-3038
21-bp repeats: 3042-3062, 3063-3083 & 3085-3105
Early promoter element: 3118-3124
Major transcription start points: 3114, 3152, 3158 & 3163
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3246-3248
Stop codon: 4038-4040
G->A mutation to remove Pst I site: 3428
C->A (Arg to Ser) mutation to remove BssH II site: 3774
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4276-4281 & 4289-4294
pUC plasmid replication origin: 4625-5268