pCoofy44
(Plasmid #55185)

• Purpose: (Empty Backbone) Insect expression vector for parallel SLIC cloning containing N-terminal 10x His and SumoStar tags. C-terminal tags can be added during SLIC.
• Depositing Lab: Sabine Suppmann
• Publication: Scholz et al BMC Biotechnol. 2013 Feb 14;13(1):12.

Backbone

• Vector backbone: pFastBac
• Backbone manufacturer: Life Technologies
• Backbone size (bp): 6383
• Vector type: Bacterial Expression
• Promoter: pPH (Polyhedrin promoter)
• Tags / Fusion Proteins:
  • 10x His (N terminal on backbone)
  • SUMOStar (N terminal on backbone)
  • PreScission site (3C protease site) (N terminal on backbone)
  • 10x His-Stop-OneStrep-Stop-S-tag-Stop-HPC4-Stop-CPD54 (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DB3.1
• Growth instructions: ccdB Survival cells are NOT suitable for the unmodified plasmid containing the ccdB gene. The plasmid is provided in the recommended DB3.1 strain.
• Copy number: High Copy

Cloning Information

• 5′ sequencing primer: Polyhedrin-F
• 3′ sequencing primer: SV40pA-R

Terms and Licenses

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

Depositor Comments

C-terminal tags are separated by a stop codon. Depending on the LP2 primer used in SLIC, the desired C-terminal tag(s) can be fused to the target gene.

Use the one of the following linearization primers pairs for SLIC cloning. Choose one LP1 forward vector primer and one LP2 reverse vector primer:

LP1 forward vector primer:
SLIC Primer (3C site) 5' GGGCCCCTGGAACAGAACTTCCAG 3'

LP2 - select an LP2 primer below depending on which C-terminal tags you want to include fused to your gene of interest:
none SLIC Primer 5' CGCCATTAACCTGATGTTCTGGGG 3'
10His- SLIC Primer 5`GAGCATCATCATCATCACCAC 3'
StrepOne - SLIC Primer 5`AGCGCTTGGAGCCACCCGCAG 3'
S-Tag - SLIC Primer 5`AAAGAAACCGCTGCTGCTAAATTCG 3'
HPC4 - SLIC Primer 5`GAGGACCAGGTGGACCCCCGG 3'
Æ54CPD - SLIC Primer 5`GGCAGCGGCAAGATCCTGCAC 3'

Test each preparation of the plasmid by transforming it into non-resistant cells (ex. DH5a) to ensure negative selection by ccdB kills all the transformed cells as expected.