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Vivid Colors Fluorescent protein vectors
TABLE OF CONTENTS
PRODUCT DESCRIPTION
pRSET/CFP, pRSET/EmGFP and pRSET/BFP Vectors
Vivid Colors pcDNA6.2/EmGFP- and YFP-DEST Gateway Vectors
Vivid Colors pcDNA6.2/EmGFP and YFP-GW/TOPO Mammalian Expression Vector Kits
Vivid Colors pcDNA6.2/EmGFP-Bsd/V5-DEST Mammalian Expression Vector Kit
Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
General information
Amino acid mutations, excitation, emission wavelengths and fluorescent spectra
Other GFPs: comparison
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
PRODUCT DESCRIPTION
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pRSET/CFP, pRSET/EmGFP and pRSET/BFP Vectors
Vivid Colors pcDNA6.2/EmGFP- and YFP-DEST Gateway Vectors
Vivid Colors pcDNA6.2/EmGFP and YFP-GW/TOPO Mammalian Expression Vector Kits
Vivid Colors pcDNA6.2/EmGFP-Bsd/V5-DEST Mammalian Expression Vector Kit
Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector
pRSET/CFP, pRSET/EmGFP and pRSET/BFP Vectors
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Bacterial expression vectors that contain sequences encoding Fluorescent Proteins (FPs). These FPs are derived
from Aequorea victoria jellyfish Green Fluorescent Protein (GFP) with amino acid substitutions that alter the
spectral properties of the proteins. Upon excitation, these FPs emit a fluorescent signal corresponding to the colors
cyan (CFP), emerald green (EmGFP) and blue (BFP).
The FP sequences have been cloned into the bacterial expression vector pRSET A to produce pRSET/CFP,
pRSET/EmGFP and pRSET/BFP which serve as source vectors of the fluorescent proteins for expression in E.coli
or to move these proteins into an expression vector of choice.
These three clones will be available as made-to-order products so the turn-around-time on orders will be
approximately 5 days for these products in the US, 7 days in Canada, 3 weeks in Europe and Asia Pacific. These are
not stocked items.
Vivid Colors pcDNA6.2/EmGFP- and YFP-DEST Gateway Vectors
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Mammalian Gateway-adapted Destination vectors encoding EmGFP (Emerald Green Fluorescent Protein) or YFP
(Yellow Fluorescent Protein) as N-terminal or C-terminal tags.
EmGFP and YFP are derived from Aequorea victoria jellyfish Green Fluorescent protein (GFP) with amino acid
substitutions that alter the spectral properties of the proteins.
Each Gateway Destination Vector is supplied with either an N- or C-terminal tagged EmGFP or YFP/GW/CAT
plasmid that serves as a control for transfection of the expression clone into the target cells, as well as a control for
expression of the gene of interest.
Vivid Colors pcDNA6.2/EmGFP and YFP-GW/TOPO Mammalian Expression Vector Kits
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These kits contain mammalian TOPO-adapted Gateway expression vectors for TOPO cloning and expression of
PCR products fused to EmGFP (Emerald Green Fluorescent Protein) or YFP (Yellow Fluorescent Protein) as N-
terminal or C-terminal tags.
EmGFP and YFP are derived from Aequorea victoria jellyfish Green Fluorescent protein (GFP) with amino acid
substitutions that alter the spectral properties of the proteins.
Each kit is supplied with either an N- or C-terminal tagged EmGFP or YFP/GW/CAT plasmid that serves as a
control for transfection of the expression clone into the target cells, as well as a control for expression of the gene of
interest.
Vivid Colors pcDNA6.2/EmGFP-Bsd/V5-DEST Mammalian Expression Vector Kit
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Mammalian Gateway-adapted Destination vector for expression of the gene of interest with a C-terminal V5 epitope
tag. The vector has a separate transcriptional unit , the murine PGK (phosphoglycerate kinase-1) promoter driving
expression of EmGFP (Emerald Green Fluorescent Protein) fused to the Blasticidin resistance gene. This allows
non-invasive monitoring of transfection efficiency by fluorescence microscopy as well as selection of stable cell
lines.
The EmGFP is derived from Aequorea victoria jellyfish Green Fluorescent protein (GFP) with amino acid
substitutions that alter the spectral properties of the protein.
The Gateway Destination vector is supplied with an expression control plasmid that serves as a control for
transfection of the expression clone into the target cells, as well as a control for expression of the gene of interest.
Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector
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Lentiviral expression plasmid containing EmGFP (Emerald Green Fluorescent Protein). The EmGFP is derived
from Aequorea victoria jellyfish Green Fluorescent protein (GFP) with amino acid substitutions that alter the
spectral properties of the protein.
Designed to be used with the Virapower or Virapower II Lentiviral Expression System as a positive control for
optimization of transfection, lentiviral titer and transduction.
SHIPPING CONDITIONS
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Lyophilized vectors are shipped at room temperature.
STORAGE CONDITIONS
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Upon receipt, store vector at 鈥?20掳C.
STABILITY
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The vectors are guaranteed stable for 6 months when stored properly.
QC SPECIFICATIONS
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All vectors have been sequence verified.
The functionality of each Gateway destination vector is qualified in an LR recombination assay using Gateway庐 LR
Clonase鈩? II Enzyme Mix. The ccdB gene is assayed by transformation using an appropriate E. coli strain.
The TOPO-adapted vectors are functionally qualified using the control reagents provided in the kit.
PROTOCOL AND APPLICATION NOTES
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General information
Amino acid mutations, excitation, emission wavelengths and fluorescent spectra
Other GFPs: comparison
General information
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EmGFP, CFP, BFP and YFP can be visualized using standard FITC filters and settings.
All of these proteins (EmGFP, CFP, BFP and YFP) have been humanized for optimal mammalian expression.
Dual fluorescence studies or co-localization experiments are best done using CFP in combination with YFP. The
excitation and emission spectra for other combinations of FPs are too close to discern one from the other.
Fluorescent protein expressing cells can be fixed using 4% paraformaldehyde in PBS for 10 minutes followed by
one quick PBS rinse and 3 x 5 minute washes using 1 ml PBS.
Photobleaching (the irreversible photochemical alteration that causes the fluorescent protein to no longer fluoresce
without degrading or destroying the protein) can be decreased by using Prolong Anti-fade mounting media from
Molecular Probes (1).
Background fluorescence can be decreased by rinsing cells in PBS prior to visualization and by using glass slides.
Fluorescence can be quenched by washing cells with 0.1% sodium borohydride in PBS for 30 minutes after fixing
the cells.
Amino acid mutations, excitation, emission wavelengths and fluorescence spectra
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EmGFP
Amino Acid mutations: S65T, S72A, N149K, M153T, I167T (and F64L, this mutation is present in EmGFP in
the pRSET vector but not in the EmGFPs in any of the other vectors). This mutation was proposed to render
better folding properties to the protein at 37C, thus enhancing fluorescence at 37C. However, in-house studies
have shown that this mutation is not necessary, hence it is not present in the EmGFPs in the pcDNA6.2 DEST,
pcDNA6.2 GW/TOPO or plenti6.2 GW control vectors.
Excitation: 487 nm Emission: 509 nm
Omega Filter set: XF100
Fluorescence Spectra of Emerald GFP
100
Normalized fluorescence
80
intensity
60
40
20
0
350 400 450 500 550 600
Wavelength (nm)
CFP
Amino Acid mutations: K26R, Y66W, N146I, M153T, V163A and N164H
Excitation: 452 nm Emission: 505 nm
Omega Filter set: XF114
Chroma Filter set: 31044
Fluorescence Spectra of CFP
100
Normalized Fluorescence
80
Intensity
60
40
20
0
350 400 450 500 550 600
Wavelength (nm)
BFP
Amino Acid mutations: F64L, Y66H, Y145F, V163A and N198S
Excitation: 308-383 nm Emission: 440-447 nm
Omega Filter set: XF10
Chroma Filter set: 31021
YFP
Amino Acid mutations: S65G, S72A, K79R and T203Y
Excitation: 514 nm Emission: 527 nm
Omega Filter set: XF1042
Chroma Filter set: 41028
Fluorescence Spectra of YFP
100
Normalized Fluorescence
80
Intensity
60
40
20
0
400 450 500 550 600 650
Wavelength (nm)
Other GFPs: Cycle 3 GFP, EGFP and EmGFP
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EmGFP is the next-generation variant of EGFP where EmGFP has been further optimized for mammalian
expression. Also, EmGFP has been mutated at amino acid positions S72A, N149K, M153T and I167T which
prevents dimerization. Both EmGFP and EGFP can be visualized using the same filter sets (FITC) and settings.
When used with the recommended filter sets and settings, Cycle 3 GFP is as bright as EmGFP or EGFP. However,
when used with FITC filter sets and settings, Cycle 3 GFP is not as bright as EmGFP or EGFP. For Cycle 3 GFP,
excitation and emission maxima are 395 nm and 478 nm for primary and secondary excitation, respectively, and 507
nm for emission. For EGFP, excitation and emission maxima are 488 nm and 507-509 nm respectively.
PRODUCT DOCUMENTATION
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Manuals MSDS COA
Brochures FAQ鈥檚 Citations
Newsletters Licensing Cell lines
Vector Data
REFERENCES
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1. White J. et al, Trends in Cell Biology 1999, 9: 61-65
PRODUCT NAMES AND CATALOG NUMBERS
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Name Features Catalog Number
pRSET/CFP Vector T7 promoter, CFP insert V352-20, V35220 (made to order)
pRSET/EmGFP Vector T7 promoter, EmGFP insert V353-20, V35320 (made to order)
pRSET/BFP Vector T7 promoter, BFP insert V354-20, V35420 (made to order)
Vivid Colors pcDNA6.2/C- Gateway attR sites, CMV promoter, C- V355-20, V35520
EmGFP-DEST Gateway Vector term EmGFP, Blasticidin selection, CAT
expression control
Vivid Colors pcDNA6.2/N- Gateway attR sites, CMV promoter, N- V356-20, V35620
EmGFP-DEST Gateway Vector term EmGFP, C-term V5, Blasticidin
selection, CAT expression control
Vivid Colors pcDNA6.2/C-YFP- Gateway attR sites, CMV promoter, C- V357-20, V35720
DEST Gateway Vector term YFP, Blasticidin selection, CAT
expression control
Vivid Colors pcDNA6.2/N-YFP- Gateway attR sites, CMV promoter, N- V358-20, V35820
DEST Gateway Vector term YFP, C-term V5, Blasticidin
selection, CAT expression control
Vivid Colors pcDNA6.2/C- TOPO TA cloning, Gateway attB sites, K359-20, K35920
EmGFP-GW/TOPO Mammalian CMV promoter, C-term EmGFP,
Expression Vector Kit Blasticidin selection, CAT expression
control
Vivid Colors pcDNA6.2/N- TOPO TA cloning, Gateway attB sites, K360-20, K36020
EmGFP-GW/TOPO Mammalian CMV promoter, N-term EmGFP, C-term
Expression Vector Kit V5, Blasticidin selection, CAT
expression control
Vivid Colors pcDNA6.2/C-YFP- TOPO TA cloning, Gateway attB sites, K361-20, K36120
GW/TOPO Mammalian CMV promoter, C-term YFP, Blasticidin
Expression Vector Kit selection, CAT expression control
Vivid Colors pcDNA6.2/N-YFP- TOPO TA cloning, Gateway attB sites, K362-20, K36220
GW/TOPO Mammalian CMV promoter, N-term YFP, C-term V5,
Expression Vector Kit Blasticidin selection, CAT expression
control
Vivid Colors Gateway attR sites, CMV promoter, C- V366-20, V36620
pcDNA6.2/EmGFP-Bsd/V5- term V5, PGK promoter driving the
DEST Mammalian Expression EmGFP-Blasticidin fusion gene, CAT
Vector Kit expression control
Vivid Colors pLenti6.2- Gateway attB sites, EmGFP insert, V369-20, V36920
GW/EmGFP Expression Control Blasticidin selection
Vector
COMPONENTS
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Name Part Number Catalog Number Components
pRSET/CFP Vector 432013 V352-20, (made to order) 10 ug pRSET/CFP, lyophilized in TE, pH 8.0
V35220
pRSET/EmGFP Vector 432012 V353-20, (made to order) 10 ug pRSET/EmGFP, lyophilized in TE, pH
V35320 8.0
pRSET/BFP Vector 432014 V354-20, (made to order) 10 ug pRSET/BFP, lyophilized in TE, pH 8.0
V35420
Vivid Colors 431900 V355-20 6 ug pcDNA6.2/C-EmGFP-DEST Gateway
pcDNA6.2/C-EmGFP- vector, lyophilized in TE, pH 8.0
DEST Gateway Vector
10 ug pcDNA6.2/C-EmGFP/GW/CAT
control vector, lyophilized in TE, pH 8.0
Vivid Colors 431901 V356-20 6 ug pcDNA6.2/N-EmGFP-DEST Gateway
pcDNA6.2/N-EmGFP- vector, lyophilized in TE, pH 8.0
DEST Gateway Vector
10 ug pcDNA6.2/N-EmGFP/GW/CAT
control vector, lyophilized in TE, pH 8.0
Vivid Colors 431902 V357-20 6 ug Vivid Colors pcDNA6.2/C-YFP-DEST
pcDNA6.2/C-YFP- Gateway vector, lyophilized in TE, pH 8.0
DEST Gateway Vector
10 ug pcDNA6.2/C-YFP/GW/CAT control
vector, lyophilized in TE, pH 8.0
Vivid Colors 431903 V358-20 6 ug pcDNA6.2/N-YFP-DEST Gateway
pcDNA6.2/N-YFP- vector, lyophilized in TE, pH 8.0
DEST Gateway Vector
10 ug pcDNA6.2/N-YFP/GW/CAT control
vector, lyophilized in TE, pH 8.0
Vivid Colors 431908 V366-20, V36620 6 ug pcDNA6.2/EmGFP-Bsd/V5-DEST
pcDNA6.2/EmGFP- Gateway vector, lyophilized in TE, pH 8.0
Bsd/V5-DEST
Mammalian Expression 10 ug pcDNA6.2/EmGFP-Bsd/V5-GW/CAT
Vector Kit control vector, lyophilized in TE, pH 8.0
Vivid Colors pLenti6.2- 431907 V369-20, V36920 20 ug pLenti6.2-GW/EmGFP, lyophilized in
GW/EmGFP Expression TE, pH 8.0
Control Vector
Vivid Colors pcDNA6.2/C-EmGFP-GW/TOPO Mammalian Expression Vector Kit
Components Part Number Catalog Number Size
pcDNA6.2/C-EmGFP- 451900 K359-20, K35920 20 reactions
GW/TOPO cloning kit
One Shot TOP10 440301
chemically competent
E.coli
Vivid Colors pcDNA6.2/N-EmGFP-GW/TOPO Mammalian Expression Vector Kit
Components Part Number Catalog Number Size
pcDNA6.2/N-EmGFP- 451901 K360-20, K36020 20 reactions
GW/TOPO cloning kit
One Shot TOP10 440301
chemically competent
E.coli
Vivid Colors pcDNA6.2/C-YFP-GW/TOPO Mammalian Expression Vector Kit
Components Part Number Catalog Number Size
pcDNA6.2/C-YFP- 451902 K361-20, K36120 20 reactions
GW/TOPO cloning kit
One Shot TOP10 440301
chemically competent
E.coli
Vivid Colors pcDNA6.2/N-YFP-GW/TOPO Mammalian Expression Vector Kit
Components Part Number Catalog Number Size
pcDNA6.2/N-YFP- 451903 K362-20, K36220 20 reactions
GW/TOPO cloning kit
One Shot TOP10 440301
chemically competent
E.coli
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