pEGFP- C1 Plasmid

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pEGFP-C1

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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-vector

 

pEGFP-C1 Sequence

LOCUS       Exported                4731 bp ds-DNA     circular SYN 30-AUG-2016
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4731)
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, August 30, 2016 
FEATURES             Location/Qualifiers
     source          1..4731
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             613..1329
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    1330..1395
                     /note="MCS"
                     /note="multiple cloning site"
     polyA_signal    1519..1640
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1647..2102)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2129..2233
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2235..2592
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2443..2578
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2627..3421
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3653..3700
                     /note="HSV TK poly(A) signal"
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4029..4617
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga
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