pmKate2-clathrin vector

cat.# FP322

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-clathrin FP322 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 clathrin LCB

Vector description

pmKate2-clathrin is a mammalian expression vector encoding mKate2-clathrin fusion protein (see reporter description). The vector can be used for fluorescent labeling of clathrin LCB in living cells.

C-terminal mKate2 fusion construct with human clathrin light chain expressed in human cervical adenocarcinoma cells (HeLa). Scale bar represents 10 μm. Image from Shcherbo et al., 2009.

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human clathrin LCB is fused to the mKate2 C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2-clathrin coding sequence [Kozak, 1987].

pmKate2-clathrin vector can be used as a source of mKate2-clathrin 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-clathrin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-clathrin 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
mKate2-clathrin fusion: 613-1992
mKate2: 613-1314
Start codon (ATG): 613-615
Last amino acid in mKate2: 1312-1314
Clathrin LCB: 1360-1992
Stop codon: 1993-1995
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2156-2161 & 2185-2190
mRNA 3' ends: 2194 & 2206
f1 single-strand DNA origin: 2253-2708
Bacterial promoter for expression of Kan^r gene
-35 region: 2770-2775
-10 region: 2793-2798
Transcription start point: 2805
SV40 origin of replication: 3049-3184
SV40 early promoter
Enhancer (72-bp tandem repeats): 2882-2953 & 2954-3025
21-bp repeats: 3029-3049, 3050-3070 & 3072-3092
Early promoter element: 3105-3111
Major transcription start points: 3101, 3139, 3145 & 3150
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3233-3235
Stop codon: 4025-4027
G->A mutation to remove Pst I site: 3415
C->A (Arg to Ser) mutation to remove BssH II site: 3761
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4263-4268 & 4276-4281
pUC plasmid replication origin: 4612-5255