■ 基本信息
別名: | pEGFPC1 |
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: | 4731bp |
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(wú)須誘導(dǎo)，瞬時(shí)表達(dá) |
5'測(cè)序引物:
| pEGFP-C-5
|
3'測(cè)序引物: | pEGFP-C-3 |
■ 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
pEGFP-C1編碼野生型GFP的紅移型變體，其已經(jīng)針對(duì)更明亮的熒光和哺乳動(dòng)物細(xì)胞中更高的表達(dá)進(jìn)行了優(yōu)化。（激發(fā)最大= 488nm;發(fā)射最大值= 507nm）pEGFP-C1編碼GFPmut1變體，其含有Phe-64對(duì)Leu和Ser-65至Thr的雙氨基酸取代pEGFPC1中的MCS在EGFP編碼序列和SV40 polyA之間克隆到MCS中的基因如果與EGFP的閱讀框處于相同的閱讀框并且不存在中間終止密碼子，則將其表達(dá)為EGFP的C末端的融合物。 EGFP基因下游的SV40聚腺苷酸信號(hào)直接適當(dāng)處理EGFP mRNA的3'末端。載體骨架還含有用于在表達(dá)SV40 T抗原的哺乳動(dòng)物細(xì)胞中復(fù)制的SV40來(lái)源。由SV40早期啟動(dòng)子，Tn5的新霉素/卡那霉素抗性基因和來(lái)自單純皰疹病毒胸苷激酶（HSV TK）基因的多聚腺苷酸化信號(hào)組成的新霉素抗性盒（Neor）允許使用G418選擇穩(wěn)定轉(zhuǎn)染的真核細(xì)胞。該盒上游的細(xì)菌啟動(dòng)子在大腸桿菌中表達(dá)卡那霉素抗性。 pEGFP-C1backbone還提供了用于在大腸桿菌中繁殖的pUC復(fù)制起點(diǎn)和用于單鏈DNA生產(chǎn)的f1來(lái)源。 質(zhì)粒只保證關(guān)鍵序列正確，不保證表達(dá)效果。
pEGFP-C1encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm;emission maximum = 507 nm.) pEGFP-C1 encodes the GFPmut1 variant (4) 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 (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFPC1is 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-C1backbone 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-C1 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-C1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
Propagation in E. coli
? 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.
? E. coli replication origin: pUC
? Copy number: ≈500
? Plasmid incompatibility group: pMB1/ColE1
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請(qǐng)下載：
ZK414pEGFP-C1哺乳熒光質(zhì)粒.txt
