pEGFP-1启动子绿色荧光报告载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pEGFP-1启动子绿色荧光报告载体

BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

描述：

promoterless，EGFP增强型绿色荧光蛋白基因，Kan抗性-G418筛选标记

Map图谱：

Restriction Map and Multiple Cloning Site (MCS) of pEGFP-1. All restriction sites shown are unique. The Not I site follows

the EGFP stop codon. The Xba I site (*) is methylated in the DNA provided by BD Biosciences. If you wish to digest the vector

with this enzyme, you will need to transform the vector into a dam– host and make fresh DNA.

Description

pEGFP-1 encodes 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-1 encodes the GFPmut1 variant (4) which contains the double-aminoacid

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. pEGFP-1 is a promoterless EGFP

vector which can be used to monitor transcription from different promoters and promoter/enhancer

combinations inserted into the MCS located upstream of the EGFP coding sequence. 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) allows stably transfected

eukaryotic cells to be selected using G418. The Neor cassette consists 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. A bacterial promoter upstream of this cassette

confers kanamycin resistance in E. coli. The pEGFP-1 backbone also provides a pUC origin of

replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Use

EGFP can be used as an in vivo reporter of gene expression (2). Promoters should be cloned into the pEGFP-1 MCS

upstream from the EGFP coding sequences. Without the addition of a functional promoter, this vector will not express

EGFP. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection

method. If required, stable transformants can be selected using G418 (7).

Location of features

• MCS: 12–89

• Enhanced green fluorescent protein (EGFP) gene

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 814–816

Insertion of Val at position 2: 100–102

GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 289–294

His-231 to Leu mutation (A→T): 791

• SV40 early mRNA polyadenylation signal

Polyadenylation signals: 970–975 & 999–1004

mRNA 3' ends: 1008 & 1020

• f1 single-strand DNA origin: 1067–1522

(Packages noncoding strand of EGFP.)

• Ampicillin resistance (β-lactamase) promoter

–35 region: 1584–1589; –10 region: 1607–1612

Transcription start point: 1619

• SV40 origin of replication: 1863–1998

• SV40 early promoter

Enhancer (72-bp tandem repeats): 1694–1767 & 1768–1839

21-bp repeats: 1843–1863, 1864–1884 & 1886–1906

Early promoter element: 1919–1925

Major transcription start points: 1915, 1953, 1959 & 1964

• Kanamycin/neomycin resistance gene

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2047–2049; stop codon: 2839–2841

G→A mutation to remove Pst I site: 2229

C→A (Arg→Ser) mutation to remove BssH II site: 2575

• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3077–3082 & 3090–3095

• pUC plasmid replication origin: 3426–4069

Primer Locations

• EGFP-N Sequencing Primer (#6479-1): 163–142

• EGFP-C Sequencing Primer (#6478-1): 750–771

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 XL-1 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

References

1. Prasher, D. C., et al. (1992) Gene 111:229–233.

2. Chalfie, M., et al. (1994) Science 263:802–805.

3. Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 341:277–280.

4. Cormack, B., et al. (1996) Gene (in press).

5. Haas, J., et al. (1996) Curr. Biol. 6:315–324.

6. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.

7. Gorman, C. (1985). In DNA cloning: A practical approach, vol. II. Ed. D.M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

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