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產品名稱:pEGFP-N1(ZK411)

貨號 規格 價格 訂購數量 是否現貨
ZK411 1ug×1支 870 - + 有貨
質粒分類
主要功能次級功能用途抗性
哺乳細胞質粒
哺乳熒光質粒
哺乳綠色質粒
——Kan


■ 基本信息

啟動子:CMV promoter

復制子:pUC ori,f1 ori

終止子:SV40 poly(A) signal

質粒分類:哺乳系列質粒;哺乳熒光質粒;哺乳綠色質粒

質粒大小:4733bp

質粒標簽:C-EGFP

原核抗性:Kan,50μg/ml

篩選標記:Neo/G418

克隆菌株:DH5α大腸桿菌

培養條件:37℃,有氧,LB

表達宿主:293T等哺乳細胞

培養條件:37℃,有氧,5%CO2

誘導方式:無須誘導,瞬時表達

5'測序引物:pEGFP-N-5:TGGGAGGTCTATATAAGCAGAG

3'測序引物:pEGFP-N-3: CGTCGCCGTCCAGCTCGACCAG


■ 質粒簡介

pEGFP-N1 encodes a red-shifted variant of wild-type GFP  which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N1 encodes the GFPmut1 variant 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. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site  to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N1 is between the immediate early promoter of CMV and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-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, 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 transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Fusions to the N 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-N1 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 . pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).


■ 質粒圖譜


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