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to construct a new plasmid(in E. coli), what features do i need


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#1 clwop

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Posted 29 August 2009 - 05:35 AM

I want to develop a new plasmid from PET-28a.
In the map of pet-28 released by Novagen, there are mainly 5 features: col E1 pBR322 origin; Kanr; F1 origin; multiple cloning site and lac I.
I don't want the F1 origin; multiple cloning site and lac I in my new plasmid. So i plan to pcr the col E1 pBR322 origin and Kanr from pet 28 a, ligate this linear fragment with another fragment (for example a gene) to build a new plasmid.
can such a plasmid be maintained in E coli? the pBR322 origin and Kanr are necessary. but are they enough? what about the enzymes for plasmid replication? are they Encoded by the plasmid itself or by the host?



and hi everyone this is my fist post here. nice to meet u

#2 phage434

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Posted 29 August 2009 - 05:43 AM

That's plenty. You might want to look at some simpler starting plasmids. pUC19, for example, has just the col E1 origin and an amp resistance cassette. The col E1 origin can be cut down in size substantially and still be functional.




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