p3360_KanR_2p
(Plasmid #247302)

• Purpose: Production of pinocembrin in Escherichia coli, cloned by BASIC assembly, based on 3360 in Carbonell et al. 2018
• Depositing Lab: Alexandre Kuhn
• Publication: Assessment of state-of-the-art automation methods for the development of bioproduction strains (unpublished)

Backbone

• Vector backbone: pTwist_High copy
• Backbone manufacturer: Twist Bioscience
• Total vector size (bp): 9845
• Modifications to backbone: AmpR was replaced with KanR
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: AtCHI
• Alt name: Chalcone isomerase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 531
• Promoter: Ptrc with lac operon

Cloning Information for Gene/Insert 1

• Cloning method: Other
• 5′ sequencing primer: entire plasmid sequenced

Gene/Insert 2

• Gene/Insert name: AtCHS
• Alt name: chalcone synthase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1188

Cloning Information for Gene/Insert 2

• Cloning method: Other
• 5′ sequencing primer: entire plasmid sequenced

Gene/Insert 3

• Gene/Insert name: Sc4CL
• Alt name: 4-coumarate:CoA ligase
• Species: Streptomyces coelicolor, strain ATCC BAA-471/A3(2)/M145
• Insert Size (bp): 1569

Cloning Information for Gene/Insert 3

• Cloning method: Other
• 5′ sequencing primer: entire plasmid sequenced

Gene/Insert 4

• Gene/Insert name: AtPAL
• Alt name: phenylalanine ammonia-lyase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 2175

Cloning Information for Gene/Insert 4

• Cloning method: Other
• 5′ sequencing primer: entire plasmid sequenced

Resource Information

• Supplemental Documents:
  • p3360_KanR_2p.gbk
• A portion of this plasmid was derived from a plasmid made by: JBEI - ICE Inventory of composable elements, for gene sequences AtCHI, AtCHS, Sc4CL, and AtPAL. Alicia Da Silva and Jean-Loup Faulon at Micalis, INRAE, France for linkers used for the BASIC assembly.

Terms and Licenses

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

Depositor Comments

The gene sequences (AtCHI, AtCHS, Sc4CL and AtPAL were obtained from JBEI - ICE -Inventory of composable elements and the plasmids were based on the publication:

Carbonell, P., Jervis, A.J., Robinson, C.J. et al. An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals. Commun Biol 1, 66 (2018). https://doi.org/10.1038/s42003-018-0076-9

The Linkers used for the BASIC assembly were kindly provided by Alicia Da Silva and Jean-Loup Faulon at Micalis, INRAE, France.