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.
|Nhe I||Bgl II||Sac I||EcoR I||Sal I||Sac II||Sma I/Xma I||Age I||Katushka2S|
|Afe I||Xho I||Hind III||Pst I||Kpn I||Apa I||BamH I||Nco I*|
* – not unique site.
pKatushka2S-N is a mammalian expression vector encoding far-red fluorescent protein Katushka2S (see reporter description). The vector allows generation of fusions to the Katushka2S N-terminus and expression of Katushka2S fusions or Katushka2S alone in eukaryotic (mammalian) cells.
Katushka2S 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 Katushka2S coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and Katushka2S coding sequence.
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Generation of Katushka2S 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 Katushka2S N-terminus when inserted in the same reading frame as Katushka2S and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. Katushka2S-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express Katushka2S when transfected into eukaryotic (mammalian) cells.
Note: The plasmid DNA was isolated from dam+-methylated
Expression in mammalian cells
pKatushka2S-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of Katushka2S or its fusions in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1384-1386
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1539-1544 & 1568-1573
mRNA 3' ends: 1577 & 1589
f1 single-strand DNA origin: 1636-2091
Bacterial promoter for expression of Kanr gene
-35 region: 2153-2158
-10 region: 2176-2181
Transcription start point: 2188
SV40 origin of replication: 2432-2567
SV40 early promoter
Enhancer (72-bp tandem repeats): 2265-2336 & 2337-2408
21-bp repeats: 2412-2432, 2433-2453 & 2455-2475
Early promoter element: 2488-2494
Major transcription start points: 2484, 2522, 2528 & 2533
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2616-2618
Stop codon: 3408-3410
G->A mutation to remove Pst I site: 2798
C->A (Arg to Ser) mutation to remove BssH II site: 3144
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3646-3651 & 3659-3664
pUC plasmid replication origin: 3995-4638
Katushka2S-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 8,138,320; European Pat. 1994149; and other Evrogen Patents and/or Patent applications pending. By use of these Products, you accept the terms and conditions of the applicable Limited Use Label License.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839, and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
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