pEYFP-Mem细胞膜定位黄色荧光表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pEYFP-Mem细胞膜定位黄色荧光表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
Description:
pEYFP-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin,
also called GAP-43 (1), and a yellow-green fluorescent variant of the enhanced green fluorescent
protein (EGFP). The neuromodulin fragment contains a signal for posttranslational palmitoylation of
cysteines 3 and 4 that targets EYFP to membranes. The EYFP gene contains four amino acid
substitutions previously published as GFP-10C (2). The fluorescence excitation maximum of EYFP is 513
nm, and the emission maximum is 527 nm (in the yellow-green region).
In addition to the chromophore mutations, EYFP contains >190 silent mutations that create an open
reading frame comprised almost entirely of preferred human codons (3). Furthermore, upstream
sequences flanking the EYFP-Mem fusion protein have been converted to a Kozak consensus
translation initiation site (4). These changes increase the translational efficiency of the fusion protein and
consequently its expression in mammalian cells.
Expression of EYFP-Mem is driven by the immediate early promoter of CMV (PCMV IE). The vector contains
an SV40 origin of replication and a neomycin resistance (Neor) gene for selection in mammalian cells.
A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector
backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for singlestranded
DNA production.
Use:
pEYFP-Mem can be transfected into mammalian cells using any standard method. If required, stable
transformants can be selected using G418 (5). Expression of EYFP-Mem in mammalian cells results in
strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This
membrane labeling also permits study of fine cellular processes such as neuronal axons (6), leading
edges of migrating cells, filopodia, or microvilli on cell surfaces. pEYFP-Mem cannot be used as an
exclusive plasma membrane marker because it also partially labels intracellular membranes.
Location of features:
• 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
• EYFP-Mem fusion gene
Kozak consensus translation initiation site: 672–682
Start codon (ATG): 679–681
Neuromodulin N-terminal sequence: 679–738
Enhanced yellow fluorescent protein (EYFP) gene: 739–1458
Insertion of Val at position 2: 742–744
GFP-10C mutations:
Ser-65 to Gly: 934–936
Val-68 to Leu: 943–945
Ser-72 to Ala: 955–957
Thr-203 to Tyr: 1348–1350
His-231 to Leu mutation (A→T): 1433
Stop codon: 1456–1458
• SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1612–1617 & 1641–1646
mRNA 3' ends: 1650 & 1662
• f1 single-strand DNA origin: 1709–2164
(Packages the noncoding strand of EYFP-Mem.)
• Bacterial promoter for expression of Kanr gene:
–35 region: 2226–2231; –10 region: 2249–2254
Transcription start point: 2261
• SV40 origin of replication: 2505–2640
• SV40 early promoter
Enhancer (72-bp tandem repeats): 2338–2409 & 2410–2481
21-bp repeats: 2485–2505, 2506–2526 & 2528–2548
Early promoter element: 2561–2567
Major transcription start points: 2557, 2595, 2601 & 2606
• Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2689–2691; stop codon: 3481–3483
G→A mutation to remove Pst I site: 2871
C→A (Arg to Ser) mutation to remove BssH II site: 3217
• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3719–3724 & 3732–3737
• pUC plasmid replication origin: 4068–4711
Propagation in E. coli:
• Suitable host strains: DH5, HB101, and other general-purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue.
• Selectable marker: plasmid confers resistance to kanamycin (50 g/ml) in E. coli hosts.
• E. coli replication origin: pUC
• Copy number: ~500
• Plasmid incompatibility group: pMB1/ColE1
References:
1. Skene, J. H. P. & Virag, I. (1989) J. Cell. Biol. 108:613–625.
2. Ormö, M. et al. (1996) Science 273:1392–1395.
3. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
4. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.
5. Gorman, C. (1985) In DNA cloning: a practical approach, vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
6. Moriyoshi, K., et al. (1996) Neuron 16:255–260.

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