pmKate2-lyso vector

cat.# FP312

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 pmKate2-lyso FP312 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 mKate2 Reporter codon usage mammalian Promoter for mKate2 PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use far-red fluorescent labeling of lysosomes

Vector description

pmKate2-lyso is a mammalian expression vector intended for far-red fluorescent labeling of lysosomes in living cells. The vector encodes far-red fluorescent protein mKate2 (see reporter description) targeted to lysosomal membrane by rat Lysosomal Associated Membrane Protein 1 (LAMP-1), fused to the mKate2 N-terminus.

Transiently transfected HeLa cells expressing mKate2 fusion with rat Lysosomal Associated Membrane Protein 1 (LAMP-1).

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].

pmKate2-lyso vector can be used as a source of mKate2-LAMP1 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

pmKate2-lyso vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-LAMP1 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
mKate2-LAMP1 fusion
Start codon (ATG): 626-628
Lysosomal Associated Membrane Protein 1 (LAMP1) sequence: 626-1846
Start of mKate2 coding sequence (ATG): 1907-1909
Stop codon: 2603-2605
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2758-2763 & 2787-2792
mRNA 3' ends: 2796 & 2808
f1 single-strand DNA origin: 2855-3310
Bacterial promoter for expression of Kan^r gene
-35 region: 3372-3377
-10 region: 3395-3400
Transcription start point: 3407
SV40 origin of replication: 3651-3786
SV40 early promoter
Enhancer (72-bp tandem repeats): 3484-3555 & 3556-3627
21-bp repeats: 3631-3651, 3652-3672 & 3674-3694
Early promoter element: 3707-3713
Major transcription start points: 3703, 3741, 3747 & 3752
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3835-3837
Stop codon: 4627-4629
G->A mutation to remove Pst I site: 4017
C->A (Arg to Ser) mutation to remove BssH II site: 4363
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4865-4870 & 4878-4883
pUC plasmid replication origin: 5214-5857