pJ204-loxP-EF1a-tagBFP-HygR-lox2272-BGHpA-siteT9-HDR
(Plasmid #154827)

• Purpose: Landing pad for Cre/lox-based recombinase-mediated cassette exchange (RMCE) in CHO cells, donor plasmid for CRISPR/Cas9-mediated targeted integration in CHO cells (site T9)
• Depositing Lab: Lise Marie Grav
• Publication: Sergeeva et al ACS Synth Biol. 2020 Aug 24. doi: 10.1021/acssynbio.0c00322.

Backbone

• Vector backbone: pJ204
• Backbone manufacturer: DNA2.0
• Backbone size w/o insert (bp): 2703
• Modifications to backbone: Amplified for USER cloning
• Vector type: Mammalian Expression, Cre/Lox, CRISPR
• Selectable markers: Hygromycin

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: TagBFP
• Insert Size (bp): 702
• Promoter: EF1a

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: unknown

Gene/Insert 2

• Gene/Insert name: HygR
• Insert Size (bp): 1026
• Promoter: SV40 early promoter

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: unknown

Gene/Insert 3

• Gene/Insert name: ZsGreen1-DR
• Insert Size (bp): 828
• Promoter: CMV
• Tag / Fusion Protein:
  • PEST (C terminal on insert)

Cloning Information for Gene/Insert 3

• Cloning method: Unknown
• 5′ sequencing primer: unknown

Resource Information

• Supplemental Documents:
  • pl0626_cled.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This is a plasmid for CRISPR/Cas9-mediated targeted gene integration in Chinese hamster ovary (CHO) cells. It contains a landing pad for Cre/lox-based recombinase-mediated cassette exchange (RMCE) using poly(A)-trap. This plasmid is used as a donor vector together with Cas9 and sgRNA(site T9) plasmids.

The plasmid was assembled by USER cloning.

Please see previous research papers from our group showing examples of RMCE in CHO cells:
Grav LM et al., 2018 "Minimizing Clonal Variation During Mammalian Cell Line Engineering for Improved Systems Biology Data Generation". DOI: 10.1021/acssynbio.8b00140
Pristovšek N et al., 2019 "Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering". DOI: 10.1021/acssynbio.8b00453

Protocol for CRISPR/Cas9-mediated targeted integration in CHO cells:
Sergeeva D et al. 2019 "CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells". DOI: 10.1007/978-1-4939-9170-9_13