■ 基本信息

■ 質(zhì)粒屬性

■ 質(zhì)粒簡(jiǎn)介

pAcGFP1-N1質(zhì)粒是 AcGFP的衍生物，來(lái)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(duì)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(dá)效果。

■ 質(zhì)粒圖譜

■ 質(zhì)粒序列

質(zhì)粒序列請(qǐng)下載：ZK422pACGFP1-N1哺乳熒光質(zhì)粒.txt