change between neomycin and blasticidin resistance gene - (Oct/17/2005 )

Dear all
too many problems are going on. my boss said neomycin will not work on our damn chicken cells, so now I need to change or add the blasticidin resistance gene into pCINeo vector (promega)
I am going to PCR the blasticidin gene from pcDNA6 plasmid (invitrogen) and I think i need get all promoter with the blasticidin. I compare the two plasmids. there is an extra EM-7 promoter, what is this using for? expecially for Blasticidin?
and I did not find restriction site around neomycin, does anyone have done this kind of swap thing? what would you if you have this kind of condition?

all these things drive me no patience.

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i got this map for your plasmid...
it seems quite good for your purpose doesn't it?

moreover, EM7 promoter is a prokaryotic promoter used for expression of blasticidin restistance gene in bactérias. (reference here)
hope this helps
fred

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so EM-7 is for bacterial expression, that means I need not get it into my mammalian vector, right? that is fantastic. so this is wonderful. but another problem is that in pCINEO vector(promega), at the both end of neomycin, there is no restriction site, so I will not be able to cut it out, so I need to insert my PCR of blasticidin, which site is better, N- or C- of neomycin?
how to upload a file?

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hi
if i'm not wrong, you have a BamHI site "after" the polyadenylation signal following the NeoR gene.
So you can pcr your blasticidin resisance gene woth two BamHI sites on your primers.
The digest vector, dephosphorylate to decrease religation events, and clone your insert...

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You are right, there is a Bam HI site there. could be a stupid question, what this poly A do? does matter where blasticidin sit to this poly A?

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hi
i assume this poly A helps the polyadenylation of the Neo mRNA.
But as you're cloning the whole blasticidin cassette, there is no problem.
If you think poly A may be a problem, the blasticidin transcription direction should be in opposite sense as the neo one. As you're using only one RS, you will need to test the orientation. In this case, one of your primer must be the original BamHI site and the other one should be a compatible one (like BclI for ex). Then one side of your insert will not contain the BamHi site any more after the cloning, and you wll be able to check the direction of your orf by restriction analysis.
fred

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I did not get it. what do you mean after cloning, one side of my insert will not contain Bam HI?
do you mean after cloning I will get Bam Hi-my insert-BstBI-bamHI? how could I check the direction?

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sorry. I meant Bcl I sequence

un cutted vector :

XXXXXGGATCCXXXXX cutted in XXXXG GATCCXXXX

your insert : let says Bcl I Insert BamhI would be :
ZZZZZ TGATCA 12345 GGATCC ZZZZ

cutted in ZZZZZT GATCA12345G GATCZZZZ


then ligation can be :

XXXXGGATCA12345GGATCCXXXX wich have a BamHI site at the 3' part

or

XXXXGGATCC54321TGATCCXXXX wich have a BamHI site at the 5' part

Then digestion by BamH I and an other enzyme will produce different sizes in these cases.
fred

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how could BamHI site ligate to Bcl I site, i am confused now

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sorry. I meant Bcl I sequence

un cutted vector :

XXXXXGGATCCXXXXX cutted in XXXXG GATCCXXXX
XXXXXCCTAGGXXXXX XXXXCCTAG GXXXX

your insert : let says Bcl I Insert BamhI would be :
ZZZZZ TGATCA 12345 GGATCC ZZZZ
ZZZZZ ACTAGT 12345 CCATGG ZZZZ

cutted in
ZZZZZT..... GATCA12345G..... GATCZZZZ
ZZZZZACTAG. ....T12345CCTAG. ....GZZZZ

there are compatible restriction sites as GATC is a fragment shared by the two sites

ZZZZZT.... from BclI’ site can anneal and be ligated to GATCCXXXX
ZZZZZACTAG.... .... .... .... .... .... .... .... .... ..... GXXXX

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