pEGFP-C3

(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 sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-C3 is between the EGFP coding sequences and the SV40 poly A. Genes cloned into the MCS will be expressed 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 cassette (Neo r ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably t

ransfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses 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.

融资铜

Restriction Map and Multiple Cloning Site (MCS) of pEGFP-C3. All restriction sites shown are unique. The Bcl I site cannot be used 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 these enzymes, you will need to transform the vector into a dam – host and make fresh DNA.

pEGFP-C3 Vector Information PT3052-5GenBank Accession #: U57607

Catalog #6082-1

(1638) (2573)

Ase I

(597) I (601)

Eco (3850)

G I (1323)内蒙古乾坤金银精炼股份有限公司

Use:

人造美女郝璐璐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 sequences, 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 selected using G418 (7). pEGFP-C3 can also be used 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

Enhancer region: 59–465; TATA box: 554–560

Transcription start point: 583

C→G mutation to remove Sac I site: 569

•Enhanced green fluorescent protein gene

Kozak consensus translation initiation site: 606–616

Start codon (ATG): 613–615; Stop codon: 1408–1410

Insertion 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 phosphotransferase coding sequences:

赛马

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

理化指标•Herpes simplex virus (HSV) thymidine kinase (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

Propagation in E. coli:

•Suitable host strains: DH5α, HB101, and other general purpose 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

人事管理系统需求分析•Copy number: ≈500

•Plasmid incompatibility group: pMB1/ColE1

References:

1.Prasher, D. C., et al. (1992) Gene 111:229–233.

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.

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 sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by BD Biosciences Clontech. This vector has not been completely sequenced.

Notice to Purchaser

Use of BD Biosciences Clontech’s Living Colors™ products containing DNA sequences coding for m

utant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a license 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 licensed from Stanford University.

Not-For-Profit research institutes or entities are granted an automatic license with the purchase of this product for use in non-commercial internal research purposes, the terms of which are disclosed in detail in the license that accompanies the shipment of this product. Such license specifi-cally excludes the right to sell or otherwise transfer this product or its components to third parties.

For-Profit research institutes or entities must obtain a license from Amersham Biosciences. E-mail: gfp@amershambiosciences

Please contact BD Biosciences Clontech directly for any other assistance, including purchasing and technical support. All companies and institutions purchasing Living Colors™ products will be included in a quarterly report to Aurora Biosciences, as required by the BD Biosciences Clontech/Aurora Biosciences license agreement.

This product is intended to be used for research purposes only. It is not to be used for drug or diagno

stic purposes nor is it intended for human use. BD Biosciences Clontech products may not be resold, modified for resale, or used to manufacture commercial products without written approval of BD Biosciences Clontech.

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