pLM494
(Plasmid #100539)

• Purpose: CEN/ARS plasmid harboring expression cassettes for crtI, crtYB, crtE and tHMG1 of the β-carotene pathway, each flanked by loxPsym sites.
• Depositing Lab: Bernd Müller-Röber
• Publication: Hochrein et al Nat Commun. 2018 May 22;9(1):1931. doi: 10.1038/s41467-017-02208-6.

Backbone

• Vector backbone: pLM304
• Backbone size w/o insert (bp): 4846
• Total vector size (bp): 16125
• Modifications to backbone: Insertion of kanamycin expression cassette.
• Vector type: Yeast Expression, Cre/Lox, Synthetic Biology
• Selectable markers: URA3

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: GGPP synthase
• Alt name: crtE
• Species: X. dendrorhous
• Insert Size (bp): 1131
• GenBank ID: DQ016502.1
• Promoter: TIP1

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsaI (destroyed during cloning)
• 3′ cloning site: BsaI (destroyed during cloning)
• 5′ sequencing primer: GCTTGCCTAACTTTGTTC
• 3′ sequencing primer: GAATAGAGCAAAAGTTGGG

Gene/Insert 2

• Gene/Insert name: phytoene desaturase
• Alt name: crtI
• Species: X. dendrorhous
• Insert Size (bp): 1749
• GenBank ID: AY177424.1
• Promoter: PGK1

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsaI (destroyed during cloning)
• 3′ cloning site: BsaI (destroyed during cloning)
• 5′ sequencing primer: AATTGTCCGAATCGTGTG
• 3′ sequencing primer: GCGGGAAATAAGCTACAA

Gene/Insert 3

• Gene/Insert name: phytoene-beta carotene synthase
• Alt name: crtYB
• Species: X. dendrorhous
• Insert Size (bp): 2022
• GenBank ID: AY177204.1
• Promoter: TDH3

Cloning Information for Gene/Insert 3

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsaI (destroyed during cloning)
• 3′ cloning site: BsaI (destroyed during cloning)
• 5′ sequencing primer: GACGGTAGGTATTGATTG
• 3′ sequencing primer: GGGACTCAAAGCGTAAAA

Gene/Insert 4

• Gene/Insert name: HMG CoA reductase
• Alt name: tHMG1
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 1575
• GenBank ID: M22002.1
• Promoter: ZEO1

Cloning Information for Gene/Insert 4

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsaI (destroyed during cloning)
• 3′ cloning site: BsaI (destroyed during cloning)
• 5′ sequencing primer: TCAGTTTGCTTGATAGCC
• 3′ sequencing primer: GAATAGAGCAAAAGTTGGG

Resource Information

• Supplemental Documents:
  • pLM494.gbk
• A portion of this plasmid was derived from a plasmid made by: Received from Leslie Mitchell (Boeke lab).
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

Addgene NGS sequencing found the following mutations compared to the provided reference sequence: crtE with M176I, K346R, I347M, E353D mutations, crtI with L107F, H427Q mutations, and crtYB with A190T, E307K mutations. tHMG1 contains only residues 531-1054 compared to the NCBI reference seq [M22002.1]. These mutations/truncation do not affect plasmid function.