Oxford Expression Technologies - pOET Transfer Plasmids
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Information on the Oxford Expression Technologies pOET™ Tansfer Plasmids and Sequencing Primers
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Oxford Expression Technologies has release a new family of Transfer Plasmids that are designed to form the perfect complement to their existing flashBAC range of plasmids. The gene of interest undergoes homologous recombination into the virus genome (eg  flashBAC) from the transfer plasmid. Hence cloning into the transfer plasmid forms the essential first step in a baculovirus expression experiment. The new pOET Transfer Plasmids offer a number of features that make them an ideal starting point for expression work.
pOET Transfer Plasmids
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Go to Information on pOET1C and pOET2C pOET1C_6xHis and pOET2C_6xHis
Go to Information on pOET1N and pOET2N pOET1N_6xHis and pOET2N/C_6xHis
Go to Information on LIC_pOET Transfer Plasmids pOET6 BacMAM and pOET Gateway
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Compatability of pOET Transfer Plasmids with other Baculovirus Expression Systems
pOET™ Transfer Plasmids

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pOET range of Transfer Plasmids are compatible with any baculovirus system that utilises homologous recombination in insect cells including the flashBAC products from Oxford Expression Technologies (OET). Below is a list of those baculovirus expression systems which are compatable with the pOET Transfer plasmids:
 
  flashBAC (OET)
  flashBACGOLD (OET)
  flashBACULTRA (OET)
  BacMagic (Novagen)
  BacMagic2 (Novagen)
  BacVector 1000 (Novagen)
  BacVector 2000 (Novagen)
  BacVector 3000 (Novagen)
  BacPAK6 (Clontech)
  BaculoGold (Clontech)
  BaculoBright (Clontech)
  ProGreen (AbVector)
  ProFold (AbVector)
  ProEasy (AbVector)
  Sapphire (Orbigen)
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Technical Overview of pOET Transfer Plasmids
pOET range of baculovirus transfer plasmids are designed for high level expression of foreign genes under either the powerful AcMNPV polyhedrin (polh) promoter or under AcMNPV p6.9 promoter which provides for earlier expression than the polh promoter. All pOET transfer plasmids have a Col E1 origin of replication and an ampicillin resistance gene for selection in E. coli.

The plasmids are smaller than other available transfer plasmids and range in size between ca 4,500bp and ca 4,700bp, which greatly facilitates the cloning process. The polh coding sequences have been replaced by a multiple cloning site (MCS) containing multiple unique restriction sites for insertion of the foreign gene. The AcMNPV sequences flanking the MCS facilitate recombination with the virus DNA to insert the expression cassette into the polh locus. As well as Oxford Expression Technologies flashBAC platform, these plasmids can also be used with any other baculovirus system that uses homologous recombination to insert the foreign gene into the virus genome.
 
Common features of ALL pOET Transfer Plasmids:
  Small size simplifies the cloning process
  High level expression is achieved under the control of powerful promoters (either the polh promoter or the p6.9 promoter)
  Directional cloning of gene of interest into the plasmid is made possible by the presence of multiple unique restriction sites
  Col E1 origin of replication and an ampicillin resistance gene for selection in E. coli
  AcMNPV sequences flanking the multiple cloning site (MCS) facilitates recombination with the virus DNA to insert the expression cassette into the polh locus
  Perfect companion product to the flashBAC™ system
  Compatible with other baculovirus systems that use homologous recombination
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Product Product Features
pOET1™ and pOET2™

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pOET1 and pOET2 are baculovirus transfer plasmids designed for high level expression of foreign genes under the powerful AcMNPV polyhedrin (polh) promoter. pOET2 has the same MCS as pOET1, but in the reverse orientation.

pOET1 and pOET2 are a baculovirus transfer vectors designed for high level expression of foreign genes under the powerful AcMNPV polyhedrin (polh) gene promoter.  The vector is smaller (pOET1 - 4,541bp and pOET2 - 4,547bp) than other available transfer vectors, which greatly facilitates cloning steps.  It has a bacterial origin of replication and an ampicillin resistance gene for selection in E. coli. The polh coding sequences have been replaced by a multiple cloning site (MCS) containing 14 unique restriction sites for insertion of the foreign gene in the correct orientation. The AcMNPV sequences flanking the gene in the transfer vector MCS allow recombination with the viral DNA to insert the expression cassette into the polh locus of the virus genome.
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pOET3™ and pOET4™

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pOET3 and pOET4 plasmids combine the flexibility and convenience afforded by pOET1 and pOET2 with additional enhanced capabilities that complement the improvements in protein expression delivered by the flashBAC expresion vectors. pOET4 has the same MCS as pOET3, but in the reverse orientation.

pOET3 and pOET4 are a baculovirus transfer vectors designed for expression of foreign genes under the AcMNPV late p6.9 gene promoter, which is active much earlier in the virus replication cycle than the polyhedrin gene promoter used in pOET1 and pOET2.  This can be useful when expressing proteins that require extensive post-translational modification. The vector is smaller (pOET3 - 4,530bp and pOET4 - 4,536bp) than other available transfer vectors, which greatly facilitates cloning steps.  It has a bacterial Col E1 origin of replication and an ampicillin resistance gene for selection in E. coli. The polh coding sequences have been replaced by a multiple cloning site (MCS) containing 14 unique restriction sites for insertion of the foreign gene in the correct orientation. The AcMNPV sequences flanking the gene in the transfer vector MCS allow recombination with the viral DNA to insert the expression cassette into the polh locus of the virus genome.
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pOET5

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pOET5  is a dual promoter baculovirus transfer vector designed for high level expression of two foreign genes simultaneously under the powerful AcMNPV polyhedrin (polh) promoter and the very late p10 gene promoter. The promoters are in opposite orientations to minimize recombination. The vector is smaller (4,590bp) than other available transfer vectors, which greatly facilitates cloning steps. It has a bacterial Col E1 origin of replication and an ampicillin resistance gene for selection in E. coli. The polh and p10 coding sequences have each been replaced by a multiple cloning site (MCS) containing unique restriction sites for insertion of the foreign genes in the correct orientation. The AcMNPV sequences flanking the two MCS regions allow recombination with the viral DNA to insert the expression cassette into the polh locus of the virus genome.
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pOET1C_6xHis and pOET2C_6xHis

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pOET1C_6xHis and pOET2C_6xHis are baculovirus transfer plasmids designed for high level expression of foreign genes under the powerful AcMNPV polyhedrin (polh) promoter. The plasmid encodes an optional C-terminal 6xHis-Tag® fusion sequence that may be utilised. This greatly eases the purification of the recombinant protein since the 6×His-containing fusion proteins bind with high affinity to Ni-NTA Agarose. The polh sequences have been replaced by a multiple cloning site (MCS) containing unique set of restriction sites for insertion of the foreign gene in the correct orientation..
 
  Small size (pOET1C - 4,563bp and pOET2C - 4,593bp) - makes the cloning steps easy
  Both plasmids encode for an optional C-terminal 6xHis-Tag® fusion sequence
  High level expression from the polh promoter
  Directional cloning into plasmid is possible using unique restriction sites. pOET1C contains a unique set 18 sites and pOET2C contains a unique set of 19 sites
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pOET1N_6xHis and pOET2N/C_6xHis

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pOET1N_6xHis and pOET2N/C_6xHis are baculovirus transfer plasmids designed for high level expression of foreign genes under the powerful AcMNPV polyhedrin (polh) promoter.

The pOET1N_6xHis plasmid encodes for an optional N-terminal 6xHis-Tag® fusion sequence that may be utilised if the insert allows readthrough in the correct reading frame.

The pOET2N/C_6xHis plasmid encodes an N-terminal 6xHis-Tag® fusion sequence that may be utilised if the insert includes a stop codon. There is also a 6xHis-Tag®for C-terminal fusions where the start codon of the insert can be used to replace the start codon supplied in pOET2N/C_6xHis.

This presence of an N- or C-terminal 6×His-Tag® fusion sequence greatly eases the purification of the recombinant protein since the 6×His-containing fusion proteins bind with high affinity to Ni-NTA Agarose. If required, the 6×His-Tag® can be removed by incubating the fusion protein in the presence of the proteinase cleavage enzyme Thrombin. The PacI site at the end of the MCS provides translational stop codons in all three reading frames for expression of truncated proteins.
 
  Small size (pOET1N - 4,598bp and pOET2N/C - 4,626bp) - makes the cloning steps easy
    Both plasmids ecode for an optional N-terminal 6xHis-Tag® fusion sequence
  pOET2N/C also ecodes for an optional C-terminal 6xHis-Tag® fusion sequence
  6×His-Tag® can be removed by incubating the fusion protein in the presence of the proteinase cleavage enzyme Thrombin
  High level expression from the polh promoter
  The PacI site at the end of the MCS provides translational stop codons in all three reading frames for expression of truncated proteins
  Directional cloning into plasmid is possible using unique restriction sites. pOET1N contains a unique set of 23 sites and pOET2N/C contains a unique set of 21 sites
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pOET6 BacMAM and pOET Gateway

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pOET6 BacMAM is based on pOET1 backbone but contains the CMV immediate early gene promoter instead of the baculovirus polyhedron gene promoter and is thus suitable for transduction and expression in mammalian cells.
 
pOET Gateway™ vectors for flashBAC combined to make insertion of the target gene easier and quicker. The patented flashBAC technology offers the convenience of a one-step process for inserting foreign genes into Baculovirus genomes. Until now it has been necessary to produce a transfer plasmid containing the gene target using traditional ligation techniques with our pOET range of vectors.
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pOET Sequencing Primers

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The pOET sequencing primers can be used to sequence inserts cloned into the multiple cloning site (MCS) of any of the pOET range of transfer vectors. The forward primer anneals to the lef2 gene region upstream of the MCS and the reverse primer anneals to the ORF1629 gene region downstream of the MCS.
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