pDendra2-Fibrillarin vector

cat.# FP826

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 pDendra2-Fibrillarin FP826 20 μg This vector is provided as a free supplement to Evrogen Dendra2 expression vectors. Vector type mammalian expression vector Reporter Dendra2 Reporter codon usage mammalian Promoter for Dendra2 PCMV IE Host cells mammalian Selection prokaryotic - kanamycin eukaryotic - neomycin (G418) Replication prokaryotic - pUC ori eukaryotic - SV40 ori Use Expression of fibrillarin-targeted Dendra2 in mammalian cells under the control of CMV promoter; source of fibrillarin-targeted Dendra2 coding sequence

Vector description

pDendra2-Fibrillarin is a mammalian expression vector encoding Dendra2-fibrillarin fusion protein. The vector is intended for use as a positive control to estimate photoactivation conditions of Dendra2-tagged proteins.

Dendra2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of Dendra2-fibrillarin coding sequence.

The vector backbone also contains immediate early promoter of cytomegalovirus (P_{CMV IE}) for reporter 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

pDendra2-Fibrillarin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted Dendra2 in many cell types. 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
Dendra2
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 2299-2301
Last amino acid in Dendra: 1300-1302
Fibrillarin: 1336-2301
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2462-2467 & 2491-2496
mRNA 3' ends: 2500 & 2512
f1 single-strand DNA origin: 2559-3014
Eukaryotic promoter for expression of Kan^r gene
-35 region: 3076-3081
-10 region: 3099-3104
Transcription start point: 3111
SV40 origin of replication: 3355-3490
SV40 early promoter
Enhancer (72-bp tandem repeats): 3188-3259 & 3260-3331
21-bp repeats: 3335-3355, 3356-3376 & 3378-3398
Early promoter element: 3411-3417
Major transcription start points: 3407, 3445, 3451 & 3456
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3539-3541
Stop codon: 4331-4333
G->A mutation to remove Pst I site: 3721
C->A (Arg to Ser) mutation to remove BssH II site: 4067
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4569-4574 & 4582-4587
pUC plasmid replication origin: 4918-5561