Search name
pEGFP-C1,Plasmid pEGFP-C1,pEGFP-C1 vector
pEGFP-C1 Information
Promoter: CMV promoter
Replicon: F1 ori, pUC ori, SV40 ori
Terminator: SV40 poly (A) signal
Plasmid Classification: Mammalian Series Plasmids; Mammalian Fluorescent Plasmids; Mammalian Green Plasmids
Plasmid size: 4731 BP
Plasmid label: C-EGFP
Prokaryotic resistance: Kanamycin Kan (50 ug/ml)
Screening marker: neomycin Neo/G418
Cloning strains: Escherichia coli such as DH5alpha
Culture conditions: 37 C, aerobic LB
Expressing host: mammalian cells such as 293T
Culture conditions: 37 C, 5% CO2
Induction mode: no induction, transient expression
5'Sequencing Primers: pEGFP-C-5
3'Sequencing Primers: pEGFP-C-3
pEGFP-C1 Description
PEGFP-C1 encodes a red-shifted variant of wild-type GFP, which has been optimized for brighter fluorescence and higher expression in mammalian cells. (maximum excitation = 488 nm; maximum emission = 507 nm) pEGFP-C1 encodes GFPmut1 variant, which contains Phe-64 pairs of Leu and Ser-65 to Thr double amino acids instead of MCS in pEGFPC1. If the gene cloned into MCS between EGFP coding sequence and SV40 polyA is in the same reading frame as that of EGFP and there is no intermediate termination codon, it is expressed as a C-terminal fusion of EGFP. The SV40 polyadenylate signal downstream of EGFP gene directly and appropriately processes the 3'terminal of EGFP gene. The carrier skeleton also contains the source of SV40 for replication in mammalian cells expressing SV40 T antigen. Neor, composed of the early promoter of SV40, the neomycin/kanamycin resistance gene of Tn5 and the polyadenylation signal from the herpes simplex virus thymidine kinase (HSV TK) gene, allows the selection of stable transfected eukaryotic cells using G418. The bacterial promoter upstream of the box expressed kanamycin resistance in E. coli. PEGFP-C1backbone also provides a starting point for pUC replication for reproduction in E. coli and a source of F1 for single-stranded DNA production.
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
pEGFP-C1 Multiple cloning site
pEGFP-C1 Sequence
