pCAST01
(Plasmid #254944)

• Purpose: (Empty Backbone) pCAT.000-derived plasmid harboring -N inducible CAST system ready-to-clone the sgRNA. [EmR] for selection in cyanobacteria.
• Depositing Lab: Enrique Flores
• Depositing Organization: Instituto de Bioquimica Vegetal y Fotosintesis
  Located in Spain
• Publication: Sarasa-Buisan et al ISME Commun. 2026 Jul 16;6(1):ycag209. doi: 10.1093/ismeco/ycag209. eCollection 2026 Jan.

Backbone

• Vector backbone: pCAT.000
• Backbone manufacturer: Addgene (Cyanogate Kit # 1000000146 )
• Backbone size (bp): 8255
• Modifications to backbone: pCAST01 is a Level T Golden Gate–assembled plasmid for CRISPR-associated transposase (CAST) applications in cyanobacteria, based on the technology previously developed in Anabaena sp. PCC 7120 (Arévalo et al., 2024). The plasmid is built on the conjugative and replicative pCAT.000 backbone for transfer via triparental mating and stable maintenance. It encodes the CAST machinery (Cas12k and the tnsB–tnsC–tniQ operon) under the nitrogen-regulated PglnA promoter, enabling induction under nitrogen deprivation. The transposon cargo is flanked by left end (LE) and right end (RE) sequences and includes a constitutive eYFP expression cassette driven by Ptrc10 and an Erythromycin resistance cassette for selection in cyanobacteria after integration. The sgRNA module allows insertion of user-defined spacer sequences via a single LguI Golden Gate reaction and is oriented to minimize interference with transposase binding, making pCAST01 a modular, ready-to-use vector for programmable DNA insertion in cyanobacteria. References Arévalo. S, Pérez Rico. D, Abarca. D, Dijkhuizen.L, Sarasa-Buisan. C, Lindblad. P, Flores. E, Nierzwicki-Bauer. S, and Schluepmann. H ACS Synthetic Biology 2024 13 (3), 901-912 DOI: 10.1021/acssynbio.3c00583
• Vector type: CRISPR, Synthetic Biology ; CAST system, eYFP reporter, RSF1010 ori
• Selectable markers: Erythromycin for selection in cyanobacteria

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): EPI300
• Copy number: Low Copy

Cloning Information

• Cloning method: Golden Gate

Terms and Licenses

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

Depositor Comments

Plasmid DNA itself is unstable and degrades quickly over time. For long-term maintenance of this plasmid, store as a bacterial glycerol stock.

Please visit https://doi.org/10.64898/2026.02.17.706265 for the bioRxiv preprint.