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.
pHyPer-dMito is a mammalian expression vector encoding mitochondria-targeted HyPer (see reporter description). HyPer codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Duplicated mitochondrial targeting sequence (MTS) is fused to the HyPer N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995].
pHyPer-dMito vector can be used as a source of dMTS-HyPer hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, dMTS-HyPer coding sequence can be amplified by PCR.
Note: The plasmid DNA was isolated from dam+-methylated
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Expression in mammalian cells
pHyPer-dMito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted HyPer in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Start codon (ATG): 597-599
Mitochondrial localization signal 1 (MLS-1): 597-689
Mitochondrial localization signal 2 (MLS-2): 690-782
Start of HyPer coding sequence: 798-800
Stop codon: 2229-2231
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2385-2390 & 2414-2419
mRNA 3' ends: 2423 & 2435
f1 single-strand DNA origin: 2482-2937
Bacterial promoter for expression of Kanr gene
-35 region: 2999-3004
-10 region: 3022-3027
Transcription start point: 3034
SV40 origin of replication: 3278-3413
SV40 early promoter
Enhancer (72-bp tandem repeats): 3111-3182 & 3183-3254
21-bp repeats: 3258-3278, 3279-3299 & 3301-3321
Early promoter element: 3334-3340
Major transcription start points: 3330, 3368, 3374 & 3379
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3462-3464
Stop codon: 4254-4256
G->A mutation to remove Pst I site: 3644
C->A (Arg to Ser) mutation to remove BssH II site: 3990
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4492-4497 & 4505-4510
pUC plasmid replication origin: 4841-5484
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