Stable transfection of pegfpn1mog plasmid to utilize in. The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by clontech. The potential therapeutic effect of pegfp n1 mediated bmk ct against rat glioma c6 cells was assessed and. Figure 3 plasmid vector maps of pegfp n1 and pegfp n1 dta. Utrs of pegfpnutr25nt, pegfpn1no3utr, and pegfpnutr25ntmt4 inserts were generated by oligonucleotide annealing, as described. Producing a mammalian gfp expression vector containing. Molecular and functional characterization of the stressinduced. Expression and evolution of the mammalian brain gene ttyh1. Benchling works best when using a supported browser. Destruction of myelin weakens the electrical conduction of ions and thus leads to a lack of communication in the. Convergence of a sequence, monotone sequences in less formal terms, a sequence is a set with an order in the sense that there is a rst element, second element and so on. The coding sequence of the egfp gene contains more than 190 silent base changes which correspond to human codonusage preferences 5. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic.
Gfp gene was separated from pegfpn1 vector and was inserted in the back bone of pcdna3. When released from a cell, dta is nontoxic and cannot enter other cells independently without the help of diphtheria toxin b. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Construction of the mammalian expressing vector pegfpn1. Sirna vector paav95cmvegfpcytbasohnebgl, complete sequence 7,169 bp circular othergenetic ay640624. Patrick calsous lab is published in nucleic acids res. Construction of a eukaryotic expression vector pegfpc1.
So that if it is not spliced, the gfp will not express because of the the stop codon. Vector for fusing egfp to the nterminus of a partner protein. The gene of interest must be cloned into pmcherry n1 so that it is inframe with the mcherry coding sequence. Fast turnaround time with 100% sequence accuracy guarantee. Both the pegfpn1p53 vector and mar were doubledigested with noti. The pegfp actin vector expresses the egfpactin fusion protein in mammalian cells.
A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and whole genome collections from many organisms. Construction of a eukaryotic expression vector for pegfp. Provide pegfp n1 vectorplasmid map, full length sequence, antibiotic resistance, size and other information. The selfreplicating system of escherichia coli was used to construct the pegfp n1p53 vector. In all cloning experiments, the vector was cut by the appropriate restriction enzyme and run on a 1% agarose gel. The primers were joined to the restriction sites of hindiii and bamhi. For other reading frames, use pegfp c2 or pegfp c3. Research open access functional verification of the. In this study, we artificially synthesized the dta gene sequence and cloned it into pegfp n1 to generate the recombinant vector pegfp n1 dta.
The numbers of myoblasts in both the pegfp n1 and controls group were significantly lower than in the pegfp fst group, with only a few cells fusing into myotubes, as shown in fig. Oof 1 constructionofa eukaryotic expressionvector for pegfpfst and its biologicalactivity 2 in duck myoblasts 3q1 xinxin li, jiwen wang. Learn about the latest plasmid technologies and research tools. Vector for fusing egfp to the cterminus of a partner protein. We next tested whether this 25nt sequence might be sufficient for enrichment of egfp mrnas in mvs. Utr of the original construct was maintained to retain the sv40 polya addition site. Cloning vector pdeltae1sp1acmvgfp, complete sequence 8,068 bp. The pegfpn1 vector and p53 gene sequence were doubledigested with hindiii and bamhi simultaneously and then ligated overnight with t4 dna ligase at 4. The pegfp n1 backbone also provides a puc origin of replication for propagation in e. The aim of this study was to construct the eukaryotic expression vector pegfp n1hper2 and assess its expression in the human osteosarcoma cell line mg63.
This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. Improved fusion protein expression of egfp via the. Cloning vector pdeltae1sp1acmvgfp, complete sequence 8,068 bp linear dna af288620. Both the pegfp n1p53 vector and mar were doubledigested with noti.
Thomas wassmers lab contains the insert app and is published in cell mol life sci. This is a free resource for the scientific community that is compiled by addgene. In conclusion, the recombinant pegfp n1 hper2 plasmid was constructed successfully, and expressed effectively in mg63 cells. To see this sequence with restriction sites, features, and translations. The attached sequence filehas been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by clontech laboratories, inc. Hindiii and psti subcloning sites of the pegfpn1 vector clon. Generation of genemodified mice via cas9rna mediated gene targeting. The primers of p53 were designed with reference to the genbank sequence of the human tumor suppressor gene accession no. The results showed that pegfp n1 mediated bmk ct expression displayed a high activity in suppressing cell migration via mmp2.
In order to maintain mrna stability, part of the 3. For other reading frames, use pegfp n2 or pegfp n3. So that if it is not spliced, the gfp will not express. The map, notes, and annotations on this page and in the sequence map file are ed material. To study the effects of bmp2 on cell migration and to explore further the molecular mechanism, a eukaryotic expression vector pegfp c1bmp2 plasmid was constructed and transfected into cos7 cells by liposomes. This recombinant vector was then transfected into 293t cells to observe the effect of dta protein expression on enhanced green fluorescent protein egfp protein expression and the proliferation of 293t.
Tokue is a global leader in biotechnology innovation, offering great benefits and applications to the biopharmaceutical and diagnostic industries as well as for biotechnology research communities. The potential therapeutic effect of pegfp n1 mediated bmk ct against rat glioma c6 cells was assessed and its potential mechanism was elucidated. Construction of a eukaryotic expression vector for pegfpfst. A clone without pcr errors was selected for use as a mammalian ttyh1gfp expression vector and named pegfp n1 ttyh1. Receive the latest news, hot plasmids, discounts and more. The selfreplicating system of escherichia coli was used to construct the pegfpn1p53 vector. The pegfp n1hper2 was digested using psti and kpni, and then evaluated by agarose gel electrophoresis. The gene of interest must be cloned into pmcherryn1 so that it is inframe with the mcherry coding sequence. The recombinant plasmid was further sequenced to confirm its sequence by the beijing genomics institute bgi. Cloning vector pegfp c1, complete sequence, enhanced green fluorescent protein egfp and neomycin phosphotransferase genes, complete cds 4,731 bp circular dna u55763. Total mrna was extracted from human osteosarcoma mg63 cells, the human period 2 hper2 gene was.
Generation of genemodified mice via cas9rnamediated gene. Cloning vector pegfp n1, complete sequence, enhanced green fluorescent protein egfp and neomycin phosphotransferase genes, complete cds 4,733 bp circular dna u55762. Multiple sclerosis ms is a disease of the immune system. Over 99% success rate with no gene, no charge policy. The pegfp n1 p53 vector was constructed successfully and could be. Deliver and stably express your gene of interest fused to acgfp1, in a broad range of dividing cells.
What do you do if you have troubles with cloning into a plasmid vector. Have questions about your order, deposit, or a plasmid. Chicken embryo fibroblasts were transfected and expressed the reporter gene. For other reading frames, use pegfp n1 or pegfp n2. This project is supported bytokuewhich specializes in manufacturing ultrapure antibiotics for a broad spectrum of biotechnology applications as well as for the pharmaceutical industry.
Restriction map and multiple cloning site mcs of pegfpn1 vector. The aim of this study was to construct the eukaryotic expression vector pegfp n1 hper2 and assess its expression in the human osteosarcoma cell line mg63. The fluorescence intensity of huh7 cells cotransfected with plasmid pegfp n1 plus scramble sirna showed no significant difference, in comparison with plasmid pegfp n1 alone. The cmv promoter drives the expression of the dta coding sequence and egfp coding sequences in the same orf. The recom binant plasmid was further sequenced to confirm its sequence by the beijing genomics institute bgi. The protein is incorporated into growing actin filaments and allows for visualization of actincontaining subcellular structures in living and fixed cells. Highlevel antigen expression and sustained antigen. Construction of recombinant pegfpn1hper2 plasmid and its. Promoters should be cloned into the pegfp 1 mcs upstream from the egfp coding sequences. No fluorescence was visualized in the huh7 cells cotransfected with plasmid pegfp n1 and egfp sirna. Oct 15, 2012 diphtheria toxin a dta, a segment of the diphtheria toxin tox, inhibits protein synthesis in cells. The xba i site is methylated in the dna provided by clontech. The effects of the overexpressed bmp2 on the migration of cos7 cells and the underlying molecular mechanism were investigated. What do you do if you have troubles with cloning into a.
As a positive control the egfp fragment of the pegfp n1. Constructionofa eukaryotic expressionvector for pegfpfst and. The pegfp n1 hper2 was digested using psti and kpni, and then evaluated by agarose gel electrophoresis. The accurate construction of pegfp n1 hper2 was verified by double enzyme digestion and dna sequencing. Construction of the mammalian expressing vector pegfpn1p53. Construction of the pegfpn1p53mar vector and its effect on. For other reading frames, use pegfp n1 or pegfp n3. The pegfp n1 hper2 was digested using pst i and kpn i, and then evaluated by agarose gel electrophoresis. Dec 15, 2012 the results showed that pegfp n1 mediated bmk ct expression displayed a high activity in suppressing cell migration via mmp2.
Xhoi and notidigested gag pcr product was inserted to the xhoi and notidigested pegfpn1 dna to generate psivmac239gagn1 encoding the wildtype sequence. Producing a mammalian gfp expression vector containing neomycin resistance gene manizheh izadi, maryam abiri. Monitoring transfected cells without selection agents by. The pegfp n1hper2 was digested using pst i and kpn i, and then evaluated by agarose gel electrophoresis. In the sequel, we will consider only sequences of real numbers. With various cloning strategies my colleagues and i. Functional verification of the diphtheria toxin a gene in a. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and. Stable transfection of pegfp n1mog plasmid to utilize in multiple sclerosis gene therapy. The coding sequence of the egfp gene contains more than 190 silent base changes which correspond to. After transformation of li top10f strain cells with pegfp n1 vector and pcdna3.
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