■ 基本信息
啟動子: | 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: | 4726bp |
質(zhì)粒標簽: | C-GFP |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養(yǎng)條件: | 37℃，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
誘導(dǎo)方式: | 無須誘導(dǎo)，瞬時表達 |
5'測序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） |
3'測序引物: | Sv40-polyA-R (GAAATTTGTGATGCTATTGC) |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細胞 |
質(zhì)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(zhì)粒簡介
pAcGFP1-N1質(zhì)粒是 AcGFP的衍生物，來自Aequorea coerulescens。 AcGFP1已經(jīng)優(yōu)化了更亮的熒光。（激發(fā)最大值= 475nm;發(fā)射最大值= 505nm）AcGFP1基因的編碼序列含有沉默堿基變化，其對應(yīng)于人類密碼子使用偏好。
pAcGFP1-N1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm). The coding sequence of the AcGFP1 gene
contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pAcGFP1-N1 is between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding
sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of AcGFP1 if they are in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40
polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of theAcGFP1 mRNA. The vector backbone also contains an SV40 origin for replication in mammalian
cells expressing the SV40 T antigen.Aneomycin-resistance cassette (Neor), consisting of the SV40 earlypromoter,theneomycin/kanamycinresistancegeneofTn5,andpolyadenylationsignals fromthe
Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418.Abacterial promoter upstream of the gene expresses kanamycin resistance
in E. coli. The pAcGFP1-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 AcGFP1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAcGFP1-N1
so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can
be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (2). pAcGFP1-N1 can also be used simply to express
AcGFP1 in a cell line of interest (e.g., as a transfection marker).
? Human cytomegalovirus (CMV) immediate early promoter: 1–589
Enhancer region:59–465; TATA box: 554–560
Transcription start point: 583
C→G mutation to remove Sac I site: 569
? Multiple Cloning Site (MCS): 591–671
? Aequorea coerulescens Green Fluorescent Protein (AcGFP): 673–1389
Start codon (ATG): 673–675; Stop codon: 1390–1392
Insertion of Val at position 2: 676–678
Las amino acid: 1387–1389
? SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1545–1550 & 1574–1579; mRNA 3' ends: 1583 & 1595
? f1 single-strand DNA origin: 1642–2097 (Packages the noncoding strand of AcGFP)
? Bacterial promoter for expression of Kanr gene:
–35 region: 2159–2164; –10 region: 2159–2164
Transcription start point: 2154
? SV40 origin of replication: 2438–2573
? SV40 early promoter
Enhancer (72-bp tandem repeats): 2271–2342 & 2343–2414
21-bp repeats: 2418–2438, 2439–2459 & 2467–2481
Early promoter element: 2494–2500
Major transcription start points: 2490, 2528, 2534 & 2539
? Kanamycin/neomycin resistance gene:
Neomycin phosphotransferase coding sequences: start codon (ATG): 2622–2624; stop codon: 3414–3416
GA mutation to remove Pst I site: 2804
C-A (Arg to Ser) mutation to remove BssHII site: 3150
? Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3652–3657 & 3665–3670
? pUC plasmid replication origin: 4001–4644
質(zhì)粒只保證關(guān)鍵序列正確，不保證表達效果。
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK422pACGFP1-N1哺乳熒光質(zhì)粒.txt
