pCvL31-NanoLuc^opt
(Plasmid #255782)

• Purpose: Expresses NanoLuc luciferase (codon optimized for Chromera) in Chromera velia
• Depositing Lab: Ke Hu
• Publication: Tengganu et al J Cell Sci. 2026 Jan 1;139(1):jcs.264400. doi: 10.1242/jcs.264400. Epub 2026 Jan 13.

Backbone

• Vector backbone: pBluescript
• Backbone size w/o insert (bp): 2931
• Total vector size (bp): 5173
• Vector type: Bacterial Expression ; Chromera velia, Toxoplasma gondii

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Cv β-tubulin intron 1 + GAPDH Kozak + NanoLuc
• Alt name: NanoLuc luciferase
• Insert Size (bp): 628
• Mutation: codon optimized for Chromera velia
• Promoter: putative C. velia L31 promoter (1103 bp)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: pBluescript KS
• 3′ sequencing primer: pBluescript SK

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • NanoLuc Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Derived from pCvL31-mNeonGreen, a pBluescript-based vector containing the Chromera velia L31 promoter region (1103 bp; VEuPathDB: Cvel_7598), the mNeonGreen coding sequence, a 13-amino-acid linker, and the C. velia L31 3′ UTR (499 bp). To generate the backbone, pCvL31-mNeonGreen was digested with PstI, which cuts within the L31 promoter 340 bp upstream of the start codon, and AflII, which cuts immediately downstream of the mNeonGreen-linker stop codon.

Two DNA fragments were then prepared for insertion. Fragment 1 (381 bp), amplified from pCvL31-mNeonGreen, contained a 21 bp overlap with the digested backbone, 336 bp of the L31 promoter including the PstI site. Fragment 2 (672 bp), amplified from pCvGAPDH_NanoLuc^opt, contained the first intron of C. velia β-tubulin (106 bp; VEuPathDB: Cvel_33153), the CvGAPDH Kozak sequence, and a C. velia codon-optimized NanoLuc coding sequence.

Fragments 1 and 2 were fused by overlap-extension PCR and inserted into the PstI/AflII-digested pCvL31-mNeonGreen backbone by Gibson assembly. In the final construct, the β-tubulin intron is located within the 5′ UTR, with its 3′ end positioned 9 bp upstream of the start codon, immediately before the Kozak sequence (gctcagaagATG), without disrupting the ATG.