Avidty teamed with Atum (formerly DNA 2.0) to offer our customers an easy solution to AviTagging their E. coli expressed proteins.  The new vectors employ the Atum ElectraTm system for easy cloning from a PCR product.  

To minimize steric conflicts that can affect efficient biotinylation of the AviTag, Avidity's vectors offer the choice of a short flexible linker or extended, semi-rigid linker between the AviTAg and the target protein in either an N- or C-terminal presentation of the AviTag.  Our new vectors use either the T5 promoter or rhamnose  promoter.

Avidity Atum AviTag™ (p3A) vectors are sold as linearized plasmids designed to allow target genes to be cloned using the Atum Electra Vector System®.  Target gene cloning can be done as follows:
PCR amplify target gene using PCR primers, with SapI restriction endonuclease sequences on the 5' ends, add PCR product to a tube containing the p3A vector plus Electra Reagent mix (Product EKT-03), incubate 5 minutes and transform Avidity BirA overexpressing strains (CVB101 for chemical transformation, EVB100 or EVB101 for electroporation).

Your ORF must NOT contain any SapI recognition sites, as the Electra1 cloning process utilizes the type IIS enzyme SapI. 

PCR Forward primer for C- terminal AviTag:
5';-TACACGTACTTAGTCGCTGAAGCTCTTCTATG....(ORF)....

PCR Reverse Primers for C- terminal AviTag:
5';-AGGTACGAACTCGATTGACGGCTCTTCTGCC....(ORF Reverse Complement)....

PCR Forward Primer for N- Terminal AviTag:
5';-TACACGTACTTAGTCGCTGAAGCTCTTCTTCTCATG....(ORF)....

PCR Reverse Primer for N- Terminal AviTag:
5';-AGGTACGAACTCGATTGACGGCTCTTCTTTA....(ORF Reverse Complement).... 

Choice of vectors:
Avidity offers a total of 8 different expression vectors (See Table below).
T5 promoter (IPTG inducible), N- or C- terminal AviTag™, choice of short flexible linker or rigid, extended linker between target protein and AviTag™.
Rhamnose (Rha) inducible promoter, N- or C- terminal AviTag™, choice of short flexible linker or rigid, extended linker between target protein and AviTag™. 
All plasmids are Kanamycin resistant (KnR), high copy number, with a strong ribosome binding site (rbs).


Vector product name

(Order) 

Promoter

N- or C- terminal AviTag

Short,  Flexible linker(SF) or Rigid, Extended linker (RE)

p3A-T5-N-Avtg-SF

T5

N-

SF  (AviTag-flexible linker-your protein)

p3A-T5-N-Avtg-RE

T5

N-

RE (AviTag-stiff linker-your protein)

p3A-T5-C-Avtg-SF

T5

-C

SF (your protein-flexible linker-AviTag)

p3A-T5-C-Avtg-RE

T5

-C

RE (your protein-stiff linker-AviTag)

p3A-Rha-N-Avtg-SF

Rha

N-

SF (AviTag-flexible linker-your protein)

p3A-Rha-N-Avtg-RE

Rha

N-

RE (Avitag-stiff linker-your protein)

p3A-Rha-C-Avtg-SF

Rha

-C

SF (your protein-flexible linker-AviTag)

p3A-Rha-C-Avtg-RE

Rha

-C

RE (your protein-stiff linker-AviTag)

Resources

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