peNirFP-N vector

cat.# FP743

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 peNirFP-N FP743 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 NirFP Reporter codon usage mammalian Promoter for NirFP PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use NirFP expression in mammalian cells; generation of fusions to the NirFP N-terminus

Vector description

peNirFP-N is a mammalian expression vector encoding near-infrared fluorescent protein NirFP (see reporter description). The vector allows generation of fusions to the NirFP N-terminus and expression of NirFP fusions or NirFP alone in eukaryotic (mammalian) cells.

NirFP 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 the NirFP coding sequence [Kozak, 1987]. Fragments of exons 2 and 3 and intron 2 of human beta globin gene are added in the 3’ UTR of NirFP coding sequence in order to increase the protein expression level. Multiple cloning site (MCS) is located between P_{CMV IE} and NirFP coding sequence.

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.

Generation of NirFP fusion proteins

A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the NirFP N-terminus when inserted in the same reading frame as NirFP and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. NirFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express NirFP when transfected into eukaryotic (mammalian) cells.

Expression in mammalian cells

peNirFP-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of NirFP or its fusions 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
MCS: 591-671
NirFP
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1381-1383
Fragment of human beta globin (HBB) gene: 1392-2510
Last 35 bp of HBB exon 2: 1392-1426
HBB intron 2: 1427-2277
First 233 bp of HBB exon3: 2278-2510
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2652-2657 & 2681-2686
mRNA 3' ends: 2690 & 2702
f1 single-strand DNA origin: 2749-3204
Bacterial promoter for expression of Kan^r gene
-35 region: 3266-3271
-10 region: 3289-3294
Transcription start point: 3301
SV40 origin of replication: 3545-3680
SV40 early promoter
Enhancer (72-bp tandem repeats): 3378-3449 & 3450-3521
21-bp repeats: 3525-3545, 3546-3566 & 3568-3588
Early promoter element: 3601-3607
Major transcription start points: 3597, 3635, 3641 & 3646
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3729-3731
Stop codon: 4521-4523
G->A mutation to remove Pst I site: 3911
C->A (Arg to Ser) mutation to remove BssH II site: 4257
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4759-4764 & 4772-4777
pUC plasmid replication origin: 5108-5751