pFastBac TEV SNAPf Prescission TwinStrepII cloning vector with BioBrick PolyPromoter LIC Subcloning (438-SNAP-V3)
(Plasmid #55223)

• Purpose: (Empty Backbone)
• Depositing Lab: Scott Gradia
• Publication: Gradia et al Methods Enzymol. 2017;592:1-26. doi: 10.1016/bs.mie.2017.03.008. Epub 2017 May 15.

Backbone

• Vector backbone: pFastBac
• Vector type: Insect Expression
• Tags / Fusion Proteins:
  • SNAPf (C terminal on backbone)
  • TwinStrepII (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: High Copy

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: LicBac dual V1 F (5'-cctataactattccggattattcataccgtc-3')
• 3′ sequencing primer: LicBac dual V1 Rv (5'-caggttcagggggaggtgtg-3')

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

In order to increase the efficiency of subcloning with the Series-11 Macrobac plasmids we developed a new strategy the uses LIC (Ligation Independent Cloning). The problem with the Series-11 ligation mediated subcloning was that when the plasmid size approached or exceeded 20 kb we had to screen many colonies to find a positive. We developed a strategy to use LIC for subcloning. Because this method uses no ligase, the cloning background associated with re-ligation of the empty plasmid are eliminated.

438-SNAP-V3 has a TEV cleavable TwinStrepII-Prescission-SNAPf on the C-terminus.

The 438-SNAP-V3 vector use the LICv3 Forward and Reverse primers.

LICv3 Forward - 5'-TTTAAGAAGGAGATATAGTTC-3'
LICv3 Reverse - 5'-GGATTGGAAGTAGAGGTTCTC-3'

As the target plasmid size gets >20kb you may have to increase the amount of DNA you anneal and/or transform. We have found this method gives no background colonies at all and 100% of the colonies we pick are positive. The cells we transform into are XL1Blues with a competency ~6x10^7cfu/ul.

For more information, please see our website: http://qb3.berkeley.edu/qb3/macrolab/