The complete nucleotide sequence and list of the complete nucleotide sequence and list of restriction sites can be found at. The resulting vector, pgexkg, produced a fusion protein which was readily cleaved by thrombin, but the resulting protein had several glycine residues at its amino terminus. T 27480501 pgex6p1 27459701 bamh i ecor i sma i sal i xho i not i prescission protease leu glu val leu phe gln gly pro. Note that the sequence is numbered by the pbr322 convention, so the t7 expression region is reversed on the circular map. The pgex vectors have an expanded multiple cloning site mcs that contains six restriction sites. Collectively, the pgex vectors provide all three translational reading frames beginning with the ecori restriction site. Donor vector to use in the reaction for optimal efficiency, we recommend using the following amounts of attb pcr product or linearized b expression clone and donor vector in a 20 l bp recombination reaction. The pgem t and pgem t easy vector systems have been optimized using a 1. Both the reporter genes and the vector backbone, including the bla. The designed fusion gene strategy allows the purification of soluble and insoluble recombinant proteins to homogeneity with singlestep affinity. All pgwb vectors confer resistance to both kanamycin and hygromycin in plant. This vector can be used in combination with pacycduet1 cat. Map of the glutathione stransferase fusion vectors showing reading frames and main features.
Description continued the pgl4 vector family includes. Thrombin tac promoter lac o rbs bamh i ecor i sma i sal i xho i not i a m p r m c s i a c i g s t p b r 3 2 2 o r i a m p r pgex 4t1 5. Home resources plasmid files pgex vectors ge healthcare pgex vectors ge healthcare. Configurations available with the synthetic firefly luc2 photinus pyralis and renilla hrluc renilla reniformis genes, which have been codon optimized for more efficient expression in mammalian cells.
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. Map of the gst vectors showing the reading frames and main features. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. New vectors for high level expression of recombinant.
All thirteen vectors have stop codons in all three frames downstream from the multiple cloning site not depicted in this map. This is a free resource for the scientific community that is compiled by addgene. Figure 2 circular map and features for the pfbneo retroviral vector. A cterminal v5 epitope and polyhistidine 6xhis tag allow for detection using an antiv5 antibody and an antihis antibody respectively. A differentially regulated ap2erf transcription factor gene. The glutathione stransferase gst gene fusion system is an integrated system for the expression, purification and detection of fusion proteins produced in e. This products resides on a fisher scientific gsa or va contract. To support our community, tair access limits have been lifted until may 31. Terms and conditions 20042018 harvard medical school plasmid was created and is maintained by the dfhcc dna resource core at harvard medical schooldfhcc dna resource.
Gst gene fusion system pennsylvania state university. An equimolar amount of attb pcr product or linearized attb expression clone and the donor vector. Data file 28962284 aa gst gene fusion system glutathione s. Sca i 1875 ori pgemt vector 3003bp ampr apa i aat ii sph i bstz i nco i sac ii spe i not i bstz i pst i sal i nde i sac i bstx i nsi i t7 xmn i 1994 nae i 2695 lacz f1 ori 1 start 14 20 26 31 37 46 55 62 62 73 75 82 94 103 112 126 sp6 t t sca i 1890 ori pgemt easy vector 3018bp. Promega corporation2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 telephone 6082744330 fax 6082772601 printed in usa. Thrombin tac promoter lac o rbs bamh i ecor i sma i sal i xho i not i a m p r m c s i a c i g s t p b r 3 2 2 o r i a m p r pgex4t1 5. The pmalpiii vector is a derivative of pmalp2 in which the leader sequence of. New vectors for high level expression of recombinant proteins.
Terms and conditions 20042018 harvard medical school plasmid was created and is maintained by the dfhcc dna resource core at harvard medical schooldfhcc dna resource core at harvard medical school. Map of the glutathione stransferase fusion vectors showing the reading frames and main features. The pmalpiii vector is a derivative of pmalp2 in which the leader sequence of maltose binding protein mbp, male has been. A series of sitespecific proteases complements the. Pmalc5x vector 10 ug, 10 g, pmalc5x, n8108s, 101641058, each, login.
Polylinkers of pgex4t1, pmalc2, and a pet vector were replaced with the. Please note that enzymes with nonpalindromic recognition sequences are listed twice. In order to produce recombinant crmapkk1 protein, the corresponding gene was pcr. The sale of pdonr p4p1r multisite gateway threefragment vector construction kit. The n or cter minal tags are automatically fused subsequent to. What are the steps for cloning a gene fragment in the pgex 4t1 vector.
The n or cter minal tags are automatically fused subsequent to the lr reaction. Mammalian expression is driven by the human cytomegalovirus cmv. Please contact your local pharmacia subsidiary if a discrepancy is identified. Map of the glutathione stransferase fusion vectors showing. Basic vectors with no promoter that contain a multiple cloning region for cloning a promoter of choice. The designed fusion gene strategy allows the purification of soluble and insoluble recombinant proteins to homogeneity with. Eukaryotic expression vector containing the complete coding sequence of l. See appendix 2 control regions for pgex vectors for the control regions of the vectors.
The enzymes chosen are those which we believe to have been commercially available in june 1992. Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Orfs inserted into mcs1 can be sequenced using the. T, pgex6p1, pgex4t1, and pgex5x1 can directly accept and express cdna inserts isolated from. The vector pgex4t1 was not tested with each enzyme and therefore the accuracy of the table cannot be guaranteed. Locus exported 4969 bp dsdna circular syn 031220 definition bacterial vector for expressing fusion proteins with a thrombin site. Map of the glutathione stransferase fusion vectors showing the reading frames.
The adoptive transfer of tumorreactive t cells has been shown to mediate the regression of tumors. A differentially regulated ap2erf transcription factor. The same multiple cloning sites mcs in each vector ensure easy transfer of inserts. What are the steps for cloning a gene fragment in the pgex.
These combined dna sequence and map files can be opened with snapgene or the free snapgene viewer. I have designed the primers with the ecor1 and sal1 enzyme sites at 5. Mbp fusions are also easily affinity purified on amylose columns. Adoptive immunotherapy using tcr genemodified t cells is a promising strategy for producing tumor. T 27480501 pgex6p1 27459701 bamh i ecor i sma i sal i xho i not i prescission protease leu glu val leu phe gln gly pro leu gly ser pro glu phe pro gly arg leu glu arg pro his. Bacterial vector for expressing gst fusion proteins with a factor xa site. A novel expression vector pgex5t was constructed which directs the synthesis of a fusion protein with a histidinehexapeptide and glutathionestransferase at its nterminus and the recombinant protein at its cterminus inescherichia coli. The pgex vectors are designed for inducible, highlevel intracellular expression of genes or gene fragments. T, pgex6p1, pgex4t1, and pgex5x1 can directly accept and. Find msds or sds, a coa, data sheets and more information.
The expanded mcs facilitates the unidirectional cloning of cdna inserts obtained from libraries constructed using many available lambda vectors. If initial experiments with your pcr product are suboptimal, ratio optimization may be necessary. Restriction sites for pgex4t1 the following restriction site table was compiled using dnasis software for sequence analysis. Data file 28962284 aa gst gene fusion system glutathione. The pcmvscript vector is derived from a highcopynumber pucbased plasmid and is designed to allow protein expression in mammalian systems.
Bacterial vector for expressing fusion proteins with a thrombin site. The usefulness of this vector is re flected in the fact that we have had over 300 requests for the pgexkg plasmid. Even though stop codons in all three frames are not depicted in this map, all thirteen vectors have stop codons in all three frames downstream from the multiple cloning site. Some of the vectors contain the 35s promoter upstream of the cloning site while others have none. Product components and storage conditions product size cat. The vector has the pbr322 lowcopy origin of replication for minimized basal expression level. All vectors have stop codons in all three reading frames downstream from the mcs not depicted in this map. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 phone 6082744330 fax 6082772516 printed in usa.
1073 143 1368 490 1280 1598 917 1525 743 19 614 1116 1526 1142 666 971 305 1331 924 1126 419 887 164 1360 164 1338 1271 340 424 45 576 231 883 1417 1409 212 1429 874 997 402 1484 22