■ 基本信息
啟動(dòng)子: | CMV |
復(fù)制子: | pUC |
終止子: | SV40 poly(A) signal |
質(zhì)粒分類: | 哺乳系列質(zhì)粒；哺乳熒光質(zhì)粒；哺乳綠色質(zhì)粒 |
質(zhì)粒大小: | 4735bp |
質(zhì)粒標(biāo)簽: | C-EGFP |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37℃，5%CO2 |
表達(dá)宿主: | 293T等哺乳細(xì)胞 |
誘導(dǎo)方式: | 無須誘導(dǎo)，瞬時(shí)表達(dá) |
5'測(cè)序引物: | pEGFP-C-5\'（CATGGTCCTGCTGGAGTTCGTG） |
3'測(cè)序引物: | pEGFP-C-3\'(TATGGCTGATTATGATCAGT) |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細(xì)胞 |
質(zhì)粒用途: | 蛋白表達(dá) |
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(biāo)記: |
|
■ 質(zhì)粒簡(jiǎn)介
已經(jīng)優(yōu)化了編碼野生型GFP的紅移變體EGFP具有更亮的熒光，在哺乳動(dòng)物細(xì)胞中表達(dá)較高（最大激發(fā)波長(zhǎng)= 488nm，最大釋放波長(zhǎng)= 507nm）。 EGFP基因編碼序列，含有190多個(gè)沉默堿基變化，符合人類密碼子使用偏好。EGFP序列的一側(cè)已經(jīng)轉(zhuǎn)化為KozaK的一致的翻譯起始位點(diǎn)，這進(jìn)一步提高了真核細(xì)胞中翻譯的效率。 質(zhì)粒只保證關(guān)鍵序列正確，不保證表達(dá)效果。
pEGFP-C2 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-C2 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-C2 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 (Neor), 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-C2 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 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-C2 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. pEGFP-C2 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
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.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK415pEGFP-C2哺乳熒光質(zhì)粒.txt
