|TurboFP650||BspE I||Xho I||Hind III||Pst I||Kpn I||Apa I||BamH I||STOP|
|Bgl II||Sac I||EcoR I||Sal I||Sac II||Sma I/Xma I||Xba I#|
# – sites are blocked by dam methylation. If you wish to digest the vector with these enzymes, you will need to transform the vector into a dam- host and make fresh DNA.
pTurboFP650-C is a mammalian expression vector encoding near-infrared fluorescent protein TurboFP650 (see reporter description). The vector allows generation of fusions to the TurboFP650 C-terminus and expression of TurboFP650 fusions or TurboFP650 alone in eukaryotic (mammalian) cells.
TurboFP650 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 TurboFP650 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between TurboFP650 coding sequence and SV40 polyadenylation signal (SV40 polyA).
Generation of TurboFP650 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 TurboFP650 C-terminus when inserted in the same reading frame as TurboFP650 and no in-frame stop codons are present. TurboFP650-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express TurboFP650 when transfected into eukaryotic (mammalian) cells.
Expression in mammalian cells
pTurboFP650-C vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TurboFP650 or its fusions in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 1393-1395
Last amino acid in mKate2: 1312-1314
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1535-1540 & 1564-1569
mRNA 3' ends: 1573 & 1585
f1 single-strand DNA origin: 1632-2087
Bacterial promoter for expression of Kanr gene
-35 region: 2149-2154
-10 region: 2172-2177
Transcription start point: 2184
SV40 origin of replication: 2428-2563
SV40 early promoter
Enhancer (72-bp tandem repeats): 2261-2332 & 2333-2404
21-bp repeats: 2408-2428, 2429-2449 & 2451-2471
Early promoter element: 2484-2490
Major transcription start points: 2480, 2518, 2524 & 2529
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2612-2614
Stop codon: 3404-3406
G->A mutation to remove Pst I site: 2794
C->A (Arg to Ser) mutation to remove BssH II site: 3140
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3642-3647 & 3655-3660
pUC plasmid replication origin: 3991-4634
TurboFP650-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 8,138,320; 7,972,834; 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.
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