pComb3XTT
(Plasmid #63891)

• Purpose: Backbone of pComb3X containing TT Fab (light and heavy chains) against tetanus toxin, used as phage display/expression control and PCR template for human antibody constant regions (kappa and CH1)
• Depositing Lab: Carlos Barbas
• Publication: Andris-Widhopf et al J Immunol Methods. 2000 Aug 28;242(1-2):159-81.

Backbone

• Vector backbone: pComb3X
• Backbone size w/o insert (bp): 3300
• Total vector size (bp): 4700
• Vector type: Bacterial Expression ; phage display

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: TT Fab
• Alt name: human Fab to tetanus toxin
• Species: H. sapiens (human)
• Insert Size (bp): 1400
• Promoter: lacZ
• Tags / Fusion Proteins:
  • 6xHis (C terminal on backbone)
  • HA tag (C terminal on backbone)
  • geneIII (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SfiI (not destroyed)
• 3′ cloning site: SfiI (not destroyed)
• 5′ sequencing primer: AAG ACA GCT ATC GCG ATT GCA G
• 3′ sequencing primer: GCC CCC TTA TTA GCG TTT GCC ATC

Resource Information

• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

pComb3X is the newest of the pComb vectors. Improvements over pComb3 include increased stability and introduction of an asymmetric SfiI cassette for directional cloning of full Fab, scFv, peptide and other protein for phage display. 6xHis and HA tags allow for purification and detection. An amber stop codon was introduced to turn-off expression of the pIII fusion protein by switching to a non-supressor strain of E. coli allowing production of soluble protein without subcloning. Alternatively, the gene for phage protein pIII can be removed by SpeI/NheI digest. The “TT” refers to the human Fab to tetanus toxin which can be used as a template to amplify the kappa light chain constant region and Fd for construction of chimeric antibody libraries as described in:

Barbas, C. F., III; Burton, D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York

It can also be used as a phage display/protein expression control.