pTagFP635-laminB1 vector

cat.# FP387

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 pTagFP635-laminB1 FP387 20 μg € 400.00 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 TagFP635 Reporter codon usage mammalian Promoter for TagFP635 PCMV IE Host cells mammalian Selection prokaryotic - kanamycin eukaryotic - neomycin (G418) Replication prokaryotic - pUC ori eukaryotic - SV40 ori Use far-red fluorescent labeling of lamin B1

Vector description

pTagFP635-laminB1 is a mammalian expression vector encoding TagFP635-laminB1 fusion protein (see TagFP635 description). The vector can be used for fluorescent labeling of lamin B1 in living cells.

TagFP635 codon usage is optimized for high expression in mammalian cells, i.e. humanized (Haas et al., 1996). Human lamin B1 is fused to the TagFP635 C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of TagFP635-laminB1 coding sequence [Kozak, 1987].

pTagFP635-laminB1 can be used as a source of TagFP635-laminB1 hybrid sequence. The vector backbone contains unique restriction sites that permit it excision and further insertion into expression vector of choice.

The vector backbone also 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

pTagFP635-laminB1 can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagFP635-laminB1 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
Kozak consensus translation initiation site: 606-616
TagFP635
Start codon (ATG): 613-615
Last amino acid in TagFP635: 1321-1323
Lamin B1: 1354-3114
Stop codon: 3112-3114
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 3275-3280 & 3304-3309
mRNA 3' ends: 3313 & 3325
f1 single-strand DNA origin: 3372-3827
Bacterial promoter for expression of Kan^r gene
-35 region: 3889-3894
-10 region: 3912-3917
Transcription start point: 3924
SV40 origin of replication: 4168-4303
SV40 early promoter
Enhancer (72-bp tandem repeats): 4001-4072 & 4073-4144
21-bp repeats: 4148-4168, 4169-4189 & 4191-4211
Early promoter element: 4224-4230
Major transcription start points: 4220, 4258, 4264 & 4269
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 4352-4354
Stop codon: 5144-5146
G->A mutation to remove Pst I site: 4534
C->A (Arg to Ser) mutation to remove BssH II site: 4880
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 5382-5387 & 5395-5400
pUC plasmid replication origin: 5731-6374