■ 基本信息
啟動子: | CMV |
復制子: | pUC |
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: | 4737bp |
質粒標簽: | C-EGFP |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37℃，有氧，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
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 |
■ 質粒屬性
質粒宿主: | 哺乳細胞 |
質粒用途: | 蛋白表達 |
片段類型: | ORF
|
片段物種: | 空載體
|
原核抗性: | Kan |
真核抗性: | G418 |
熒光標記: |
|
■ 質粒簡介
pEGFP-N2 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).
質粒只保證關鍵序列正確，不保證表達效果。
■ 質粒圖譜
■ 質粒序列
質粒序列請下載：
ZK412pEGFP-N2哺乳熒光質粒.txt
