pFusionRed-PDHA1 vector

cat.# FP430

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-PDHA1 FP430 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 PDHA1

Vector description

pFusionRed-PDHA1 is a mammalian expression vector encoding FusionRed-PDHA1 fusion protein (see reporter description). The vector can be used for fluorescent labeling of PDHA1 in living cells.

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(A) – FoLu cell expressing fusion of human pyruvate dehydrogenase PDHA1 with FusionRed. The fusion protein is localized in the mitochondria. (B) – HeLa cell expressing fusion of human pyruvate dehydrogenase PDHA1 with FusionRed. The fusion protein is localized in the mitochondria. 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]. Human pyruvate dehydrogenase alpha 1 (PDHA1) is fused to the FusionRed N-terminus.

pFusionRed-PDHA1 vector can be used as a source of FusionRed-PDHA1 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-PDHA1 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-PDHA1 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
PDHA1-FusionRed fusion: 606-2504
PDHA1: 606-1772
Start codon (ATG): 606-608
Last amino acid in PDHA1: 1770-1772
FusionRed: 1806-2504
Stop codon: 2502-2504
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2657-2662 & 2686-2691
mRNA 3' ends: 2695 & 2707
f1 single-strand DNA origin: 2754-3209
Bacterial promoter for expression of Kan^r gene
-35 region: 3271-3276
-10 region: 3294-3299
Transcription start point: 3306
SV40 origin of replication: 3550-3685
SV40 early promoter
Enhancer (72-bp tandem repeats): 3383-3454 & 3455-3526
21-bp repeats: 3530-3550, 3551-3571 & 3573-3593
Early promoter element: 3606-3612
Major transcription start points: 3602, 3640, 3646 & 3651
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3734-3736
Stop codon: 4526-4528
G->A mutation to remove Pst I site: 3916
C->A (Arg to Ser) mutation to remove BssH II site: 4262
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4764-4769 & 4777-4782
pUC plasmid replication origin: 5113-5756