pCDNA3.1- EGFP

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  • Model: PVT10754
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pCDNA3.1-EGFP

 

PVT10754     2ug

 

pCDNA3.1-EGFP Description

pCDNA3.1-EGFP is a Mammalian fluorescent plasmid

 

Promoter: CMV promoter

Replicon: pUC ori, F1 ori

Terminator: BGH poly (A) signal

Plasmid classification: lactation serial plasmid; lactation expression plasmid; pCDNA series plasmid.

Plasmid size: 6173bp

Plasmid tagging: C-EGFP

Prokaryotic resistance: ampicillin Amp (100 g/ml)

Screening marker: neomycin Neo/G418

Cloning strains: E. coli DH5 and E.

Culture conditions: 37 C, aerobic, LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 C, 5%CO2

Induction mode: no need to induce

5'sequencing primers: pCDNA3.1-F (CTAGAGAACCCACTGCTTAC)

3'sequencing primers: pCDNA3.1-R (TAGAAGGCACAGTCGAGG)

 

 

pCDNA3.1-EGFP  was obtained by cloning the EGFP gene into the pCDNA3.1 vector. It was designed for stable and transient expression in mammalian hosts. Most mammalian cells can perform high levels of stable and non replicating transient expression. Human cytomegalovirus immediate early (CMV) promoter is used for high level expression in a wide range of mammalian cells. Abnormal replication in cell lines with latent infection of SV40 or expression of SV40 large T antigen (e.g. COS-1, COS-7).

The GFP in pcDNA3.1-eGFP stands for green fluorescent protein. Deoxyribonucleic acids (DNAs) are typically made up of protein, so it makes sense for a special protein to make up a specific type of DNA add-gene such as the pcDNA3.1.although other means besides cloning would also be openly discussed. In this study, a detailed overview of how pcDNA-eGFP can be produced through cloning is done thanks to the use of secondary sources; a proper review of the pcDNA3.1-eGFP is first carried out to understand this compound. Afterward, the importance of a vector in the cloning process is looked at in detail and finally the cloning system is covered extensivel GFP proteins are generally composed of 238 amino acids with molecular masses of around 26.9 KD [1] and are typically used in the field of molecular biology as one of the many reporter genes. Basically, a reporter gene is a type of gene that researchers, especially in laboratory experiments, use to attach to a pre-specified sequence of the gene (oftentimes an experimental one) such as that of bacteria, plants, animals, and cell cultures. Some of the criteria that researchers use when selecting a reporter gene include, but may not be limited to, having easily identifiable and selectable markers and their ability to introduce changes that tend to be easily spotted under certain conditions. When conducting laboratory experiments in molecular biology, it would be important for researchers to know the different variables involved and the impact that they create on the experimental environment. Therefore, it makes sense to select reporter genes that possess these qualities such as the GFP [1]. In previously published studies involving the use of GFPs, including but not limited to pcDNA3.1, it has been common for researchers to introduce the GFP gene into cells using vector-based systems. In some cases, the researchers also used recombinant viruses (attaching the GFP to them). Being used as a reporter protein, the location of the target protein can be easily identified and expressed. However, in many of the laboratory experiments, the selection market of the GFPs used was not specific enough and there is often no selection market to normalize the transfection among other reactions.

 

pCDNA3.1-EGFP Multiple cloning site

pCDNA3.1-EGFP-vector

 

pCDNA3.1-EGFP Sequence

LOCUS       Exported                6173 bp ds-DNA     circular SYN 07-12-2015
DEFINITION  synthetic circular DNA
KEYWORDS    pCDNA3.1(+)-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6173)
  TITLE     Direct Submission
  JOURNAL   Exported 2016-9-18 
FEATURES             Location/Qualifiers
     source          1..6173
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        236..615
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        616..819
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        864..882
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1003..1722
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1774..1998
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2044..2472
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2486..2815
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2666..2801
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2882..3676
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3850..3971
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4020..4036)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    4044..4060
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4068..4098)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    4113..4134
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4422..5007)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(5178..6038)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
              &nbs

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