2022年PEGFP-C质粒图谱 .pdf

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1、United States/Canada800.662.2566Asia Paci?c+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.543.61 16Clontech Laboratories,Inc.A Takara Bio Company1290 Terra Bella Ave.Mountain View,CA 94043Technical Support(US)E-mail:VectorInformation(PR29971;published 03 October 2002)Restriction Map and Multi

2、ple Cloning Site(MCS)of pEGFP-C1.All restriction sites shown are unique.The Xba I andBcl I sites(*)are methylated in the DNA provided by BD Biosciences Clontech.If you wish to digest the vector with theseenzymes,you will need to transform the vector into a dam host and make fresh DNA.DescriptionpEGF

3、P-C1 encodes a red-shifted variant of wild-type GFP(13)which has been optimized forbrighter fluorescence and higher expression in mammalian cells.(Excitation maximum=488 nm;emission maximum=507 nm.)pEGFP-C1 encodes the GFPmut1 variant(4)which contains thedouble-amino-acid substitution of Phe-64 to L

4、eu 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 euka

5、ryotic cells.The MCS in pEGFP-C1 is between the EGFP coding sequences and the SV40 poly A.Genes cloned into the MCS willbe expressed as fusions to the C-terminus of EGFP if they are in the same reading frame as EGFPand there are no intervening stop codons.SV40 polyadenylation signals downstream of t

6、he EGFPgene direct proper processing of the 3 end of the EGFP mRNA.The vector backbone also containsan SV40 origin for replication in mammalian cells expressing the SV40 T-antigen.A neomycinresistance cassette(Neor),consisting of the SV40 early promoter,the neomycin/kanamycinresistance gene of Tn5,a

7、nd polyadenylation signals from the Herpes simplex virus thymidine kinase(HSV TK)gene,allows stably transfected eukaryotic cells to be selected using G418.A bacterialpromoter upstream of this cassette expresses kanamycin resistance in E.coli.The pEGFP-C1backbone also provides a pUC origin of replica

8、tion for propagation in E.coli and an f1 origin for single-stranded DNA production.pEGFP-C1 Vector Information PT3028-5GenBank Accession#:U55763Catalog#6084-1pEGFP-C14.7 kbMlu I(1642)Dra III(1872)ApaL I (4360)Stu I(2577)Ase I(8)Nhe I(592)Eco47 III(597)Age I(601)EcoO109 I(3854)SnaB I(341)BsrG I(1323)

9、MCS(1330 1417)pUC ori HSV TKpoly Af1ori SV40poly AEGFP PCMV IEPSV40eSV40 oriP Kanr/NeorTAC AAG TCC GGA CTC AGA TCT CGA GCT CAA GCT TCG AAT TCT GCA GTC GAC GGT ACC GCG GGC CCG GGA TCC ACC GGA TCT AGA TAA CTG ATC A1340?1330?1350?1360?1370?1390?1380?1400?Hind III Xho I Apa IBsp120 I Kpn IAsp718 IBamH I

10、EGFP Xba I*Xma I Sma I Sac II Sal I Acc I Sac IEcl136 IIEcoR I Pst IBspE I Bgl IISTOPs Bcl I*名师资料总结-精品资料欢迎下载-名师精心整理-第 1 页，共 3 页 -pEGFP-C1Vector InformationBD Biosciences Clontech Protocol#PT3028-52Version#PR29971UseFusions to the C terminus of EGFP retain the fluorescent properties of the native pro

11、tein allowing the localization ofthe fusion protein in vivo.The target gene should be cloned into pEGFP-C1 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

12、method.If required,stable transformants can be selected usingG418(7).pEGFP-C1 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:1589Enhancer region:59465;TATA box:554 560Transcript

13、ion start point:583CG mutation to remove Sac I site:569?Enhanced green fluorescent protein geneKozak consensus translation initiation site:606616Start codon(ATG):613615;Stop codon:14081410Insertion of Val at position 2:616618GFPmut1 chromophore mutations(Phe-64 to Leu;Ser-65 to Thr):805810His-231 to

14、 Leu mutation(AT):1307Last amino acid in wild-type GFP:13271329?MCS:1330 1417?SV40 early mRNA polyadenylation signalPolyadenylation signals:15501555&15791584;mRNA 3 ends:1588&1600?f1 single-strand DNA origin:16472102(Packages the noncoding strand of EGFP.)?Bacterial promoter for expression of Kanr g

15、ene35 region:21642169;10 region:21872192Transcription start point:2199?SV40 origin of replication:24432578?SV40 early promoterEnhancer(72-bp tandem repeats):22762347&2348241921-bp repeats:24232443,2444 2464,&2466 2486Early promoter element:24992505Major transcription start points:2495,2533,2539&2544

16、?Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:Start codon(ATG):26272629;stop codon:34193421GA mutation to remove Pst I site:2809CA(Arg to Ser)mutation to remove BssH II site:3155?Herpes simplex virus(HSV)thymidine kinase(TK)polyadenylation signalPolyadenylation sign

17、als:36573662&36703675?pUC plasmid replication origin:40064649Primer Locations?EGFP-N Sequencing Primer(#6479-1):679658?EGFP-C Sequencing Primer(#6478-1):12661287Propagation in E.coli?Suitable host strains:DH5 ,HB101,and other general purpose strains.Single-stranded DNA production requiresa host cont

18、aining an F plasmid such as JM109 or XL1-Blue.?Selectable marker:plasmid confers resistance to kanamycin(30 g/ml)to E.coli hosts.?E.coli replication origin:pUC?Copy number:500?Plasmid incompatibility group:pMB1/ColE1References1.Prasher,D.C.,et al.(1992)Gene 111:229233.2.Chalfie,M.,et al.(1994)Scienc

19、e 263:802805.3.Inouye,S.&Tsuji,F.I.(1994)FEBS Letters 341:277280.4.Cormack,B.,et al.(1996)Gene 173:3338.5.Haas,J.,et al.(1996)Curr.Biol.6:315324.6.Kozak,M.(1987)Nucleic Acids Res.15:81258148.7.Gorman,C.(1985)In DNA Cloning:A Practical Approach,Vol.II,Ed.Glover,D.M.(IRL Press,Oxford,UK)pp.143190.名师资料

20、总结-精品资料欢迎下载-名师精心整理-第 2 页，共 3 页 -Protocol#PT3028-BD Biosciences ClontechVersion#PR299713pEGFP-C1Vector InformationNotice to PurchaserUse of BD Biosciences Clontechs Living Colors?products containing DNA sequences coding for mutant Aequorea victoria green fluorescentprotein(GFP)variants or proteins th

21、ereof 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.Not-For-Profit rese

22、arch institutes or entities are granted an automatic license with the purchase of this product for use in non-commercial internalresearch 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

23、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:Please contact BD Biosciences Clontech directly for any other assistance,including purchasing and technical support.All compani

24、es andinstitutions purchasing Living Colors?products will be included in a quarterly report to Aurora Biosciences,as required by the BD BiosciencesClontech/Aurora Biosciences license agreement.This product is intended to be used for research purposes only.It is not to be used for drug or diagnostic

25、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 CompanyNote: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.名师资料总结-精品资料欢迎下载-名师精心整理-第 3 页，共 3 页 -