(PR29969; published 03 October 2002)
Description:
pEGFP-C3 encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm;emission maximum = 507 nm.) pEGFP-C3 encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding quence of the EGFP gene contains more than 190 silent ba changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak connsus translation initiation site (6) to further increa the translation efficiency in eukaryotic cells. The MCS in pEGFP-C3 is between the EGFP coding quences and the SV40 poly A. Genes cloned into the MCS will be expresd as fusions to the C terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance castte (Neo r ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kina (HSV TK) gene, allows stably t
ransfected eukaryotic cells to be lected using G418. A bacterial promoter upstream of this castte express kanamycin resistance in E. coli . The pEGFP-C3 backbone also provides a pUC origin of replication for propagation in E. coli and an f1origin for single-stranded DNA production.
Restriction Map and Multiple Cloning Site (MCS) of pEGFP-C3. All restriction sites shown are unique. The Bcl I site cannot be ud for fusions since it contains an in-frame stop codon. The Xba I and Bcl I sites (*) are methylated in the DNA provided by BD Biosciences Clontech. If you wish to digest the vector with the enzymes, you will need to transform the vector into a dam – host and make fresh DNA.
pEGFP-C3 Vector Information PT3052-5GenBank Accession #: U57607六级分数计算
Catalog #6082-1
(1638) (2573)
A I
口罩标价超千万(597) I (601)
Eco (3850)
G I (1323)
U:
蜜雪儿英文Fusions to the C terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo. The target gene should be cloned into pEGFP-C3 so that it is in frame with the EGFP coding quences, with no intervening in-frame stop codons. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be lected using G418 (7). pEGFP-C3 can also be ud simply to express EGFP in a cell line of interest (e.g., as a transfection marker). Location of Fe
atures:
•Human cytomegalovirus (CMV) immediate early promoter: 1–589
青藏铁路英文Enhancer region: 59–465; TATA box: 554–560
Transcription start point: 583
C→G mutation to remove Sac I site: 569
奥斯卡获奖影片•Enhanced green fluorescent protein gene
acibaKozak connsus translation initiation site: 606–616
Start codon (ATG): 613–615; Stop codon: 1408–1410
Inrtion of Val at position 2: 616–618
GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 805–810
His-231 to Leu mutation (A→T): 1307
Last amino acid in wild-type GFP: 1327–1329
•MCS: 1328–1413
•SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1546–1551 & 1575–1580; mRNA 3' ends: 1584 & 1596
•f1 single-strand DNA origin: 1643–2098 (Packages the noncoding strand of EGFP)
•Bacterial promoter for expression of Kan r gene
–35 region: 2160–2165; –10 region: 2183–2188
Transcription start point: 2195
•SV40 origin of replication: 2439–2574
•SV40 early promoter
Enhancer (72-bp tandem repeats): 2272–2343 & 2344–2415
21-bp repeats: 2419–2439, 2440–2460 & 2462–2482
Early promoter element: 2495–2501
Major transcription start points: 2491, 2529, 2535 & 2540
•Kanamycin/neomycin resistance gene
Neomycin phosphotransfera coding quences:
Start codon (ATG): 2623–2625; stop codon: 3415–3417
G→A mutation to remove Pst I site: 2805
C→A (Arg to Ser) mutation to remove Bss H II site: 3151
•Herpes simplex virus (HSV) thymidine kina (TK) polyadenylation signal
Polyadenylation signals: 3653–3658 & 3666–3671
•pUC plasmid replication origin: 4002–4645
Primer Locations:
•EGFP-N Sequencing Primer (#6479-1): 679–658
•EGFP-C Sequencing Primer (#6478-1): 1266–1287
israeliPropagation in E. coli:
•Suitable host strains: DH5α, HB101, and other general purpo strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue.
•Selectable marker: plasmid confers resistance to kanamycin (30 µg/ml) to E. coli hosts.
• E. coli replication origin: pUC
•Copy number: ≈500
•Plasmid incompatibility group: pMB1/ColE1
References:
1.Prasher, D. C., et al. (1992) Gene 111:229–233.nice
2.Chalfie, M., et al. (1994) Science263:802–805.
3.Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 341:277–280.
4.Cormack, B., et al. (1996) Gene 173:33–38.nest是什么意思
5.Haas, J., et al. (1996) Curr. Biol. 6:315–324.
6.Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.
7.Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp. 143–190.
Note: The attached quence file has been compiled from information in the quence databas, published literature, and other sources, together with partial quences obtained by BD Biosciences Clontech. This vector has not been completely quenced.
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U of BD Biosciences Clontech’s Living Colors™ products containing DNA quences coding for m
utant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a licen from Amersham Biosciences under U.S. Patent Nos. 5,625,048; 5,777,079; 6,054,321 and other pending U.S. and foreign patent applications. In addition, certain BD Biosciences Clontech products are made under U.S. Patent No. 5,804,387 licend from Stanford University.
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This product is intended to be ud for rearch purpos only. It is not to be ud for drug or diagno
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© 2002, Becton, Dickinson and Company
