pEGFP-C3说明书

pEGFP-C3 Vector InformationGenBank Accession #: U57607

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Ase IApaL I (4356)(8)pUCPCMV IESnaB I (341)Nhe I (592) Eco47 III (597) Age I (601)800.662.2566+1.650.919.7300+33.(0)1.3904.6880+81.(0)77.543.6116akara Bio CompanyTerra Bella Ave.

ori EcoO109 IHSV TKEGFP (3850)poly ApEGFP-C34.7 kbBsrG I (1323) Kanr/poly ASV40 Neor(1328–1413) MCSSV40 oriori f1PSV40PeMlu I (1638)Dra III (1868)Stu(2573) I EGFP1330134013501360137013801390STOPsTAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCA CCG GAT CTA GAT AAC TGA TCA•••••••Sca I Bgl II Xho I SacHind IIIEcoR I Pst I Sal I Kpn I Apa IBamH I Xba I* Bcl I*Ecl136 II I Acc IAsp718 I SacBsp II120 I Xma Sma IIRestriction Map and Multiple Cloning Site (MCS) of pEGFP-C3. All restriction sites shown are unique. The Bcl I sitecannot be used for fusions since it contains an in-frame stop codon. The Xba I and Bcl I sites (*) are methylated in the DNAprovided by BD Biosciences Clontech. If you wish to digest the vector with these enzymes, you will need to transform thevector into a dam– host and make fresh DNA.

Description:

pEGFP-C3 encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized forbrighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm;emission maximum = 507 nm.) pEGFP-C3 encodes the GFPmut1 variant (4) which contains thedouble-amino-acid substitution of Phe- to Leu and Ser-65 to Thr. The coding sequence of theEGFP gene contains more than 190 silent base changes which correspond to human codon-usagepreferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translationinitiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS inpEGFP-C3 is between the EGFP coding sequences and the SV40 poly A. Genes cloned into theMCS will be expressed as fusions to the C terminus of EGFP if they are in the same reading frameas EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream ofthe EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone alsocontains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. Aneomycin resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virusthymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected usingG418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. ThepEGFP-C3 backbone also provides a pUC origin of replication for propagation in E. coli and an f1origin for single-stranded DNA production.

(PR29969; published 03 October 2002)

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pEGFP-C3Vector Information

Use:

Fusions to the C terminus of EGFP retain the fluorescent properties of the native protein allowing the localization ofthe fusion protein in vivo. The target gene should be cloned into pEGFP-C3 so that it is in frame with the EGFP codingsequences, with no intervening in-frame stop codons. The recombinant EGFP vector can be transfected intomammalian cells using any standard transfection method. If required, stable transformants can be selected usingG418 (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 Features:

•Human cytomegalovirus (CMV) immediate early promoter: 1–5

Enhancer region: 59–465; TATA box: 554–560Transcription start point: 583

C→G mutation to remove Sac I site: 569•Enhanced green fluorescent protein gene

Kozak consensus translation initiation site: 606–616Start codon (ATG): 613–615; Stop codon: 1408–1410Insertion of Val at position 2: 616–618

GFPmut1 chromophore mutations (Phe- to Leu; Ser-65 to Thr): 805–810His-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: 13–2098 (Packages the noncoding strand of EGFP)•Bacterial promoter for expression of Kanr gene

–35 region: 2160–2165; –10 region: 2183–2188Transcription start point: 2195

•SV40 origin of replication: 2439–2574•SV40 early promoter

Enhancer (72-bp tandem repeats): 2272–2343 & 2344–241521-bp repeats: 2419–2439, 2440–2460 & 2462–2482Early promoter element: 2495–2501

Major transcription start points: 2491, 2529, 2535 & 2540•Kanamycin/neomycin resistance gene

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2623–2625; stop codon: 3415–3417G→A mutation to remove Pst I site: 2805

C→A (Arg to Ser) mutation to remove BssH II site: 3151

•Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3653–3658 & 3666–3671•pUC plasmid replication origin: 4002–45Primer Locations:

•EGFP-N Sequencing Primer (#79-1): 679–658•EGFP-C Sequencing Primer (#78-1): 1266–1287

Propagation in E. coli:

•Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requiresa 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/ColE1References:

1.2.3.4.5.6.7.

Prasher, D. C., et al. (1992) Gene 111:229–233.Chalfie, M., et al. (1994) Science 263:802–805.

Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 341:277–280.Cormack, B., et al. (1996) Gene 173:33–38.Haas, J., et al. (1996) Curr. Biol. 6:315–324.

Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.

Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp. 143–190.

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Protocol # PT3052-5Version # PR29969

pEGFP-C3Vector Information

Note: The attached sequence file has been compiled from information in the sequence databases, publishedliterature, and other sources, together with partial sequences obtained by BD Biosciences Clontech. This vector hasnot been completely sequenced.

Notice to Purchaser

Use of BD Biosciences Clontech’s Living Colors™ products containing DNA sequences coding for mutant Aequorea victoria green fluorescentprotein (GFP) variants or proteins thereof requires a license from Amersham Biosciences under U.S. Patent Nos. 5,625,048; 5,777,079; 6,054,321and 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.

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This product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for humanuse. BD Biosciences Clontech products may not be resold, modified for resale, or used to manufacture commercial products without writtenapproval of BD Biosciences Clontech.© 2002, Becton, Dickinson and CompanyProtocol # PT3052-5Version # PR29969

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