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pLJM1-FIRE-pHLy
(Plasmid #170775)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 170775 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pLJM1-EGFP
  • Backbone manufacturer
    Addgene Plasmid #19319
  • Backbone size w/o insert (bp) 8083
  • Total vector size (bp) 10093
  • Modifications to backbone
    EGFP replaced with FIRE-pHLy pH sensor (LAMP1 signal sequence-mTFP1-Linker1-humanLAMP1-Linker2-mCherry-stop codon)
  • Vector type
    Mammalian Expression, Lentiviral
  • Selectable markers
    Puromycin

Growth in Bacteria

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

Gene/Insert

  • Gene/Insert name
    LAMP1
  • Alt name
    CD107a
  • Species
    H. sapiens (human)
  • Insert Size (bp)
    1167
  • Mutation
    84 nucleotide Signal Sequence switched to the beginning of the construct
  • GenBank ID
    NM_005561.4 NM_005561.4
  • Promoter CMV
  • Tags / Fusion Proteins
    • mTFP1 (N terminal on insert)
    • mCherry (C terminal on insert)

Cloning Information

  • Cloning method Gibson Cloning
  • 5′ sequencing primer CMV forward
  • 3′ sequencing primer hPGK promoter reverse
  • (Common Sequencing Primers)

Resource Information

  • Supplemental Documents
  • A portion of this plasmid was derived from a plasmid made by
    LAMP1 signal peptide and human LAMP1 were PCR amplified from LAMP1-mGFP (Addgene Plasmid #34831, kind gift from the Mark Von Zastrov lab, University of California, San Francisco, UCSF), mTFP1 amplified from mTFP1-pBAD (Addgene Plasmid #54553), mCherry amplified from pcDNA3.1-mCherry (Addgene Plasmid #128744). The DNA segments were PCR amplified (Phusion High-Fidelity PCR Master Mix, NEB, UK, #M0531) and fused with Gibson recombination cloning method (Gibson Assembly Master Mix, NEB, UK, #E2611) in pEGFP-N3 empty backbone. The linker sequences were incorporated into the primer sequences. The FIRE-pHLy expression cassette was cloned into lentiviral vectors with CMV promoter (pLJM1-EGFP; Addgene Plasmid #19319) by Epoch Life Science services (Sugar Land,Texas, USA).
  • Article Citing this Plasmid

Terms and Licenses

Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Chin et al. ACS Sens. 2021 Jun 8. doi: 10.1021/acssensors.0c02318.
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34102054/
Article: https://pubs.acs.org/doi/10.1021/acssensors.0c02318

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pLJM1-FIRE-pHLy was a gift from Aimee Kao (Addgene plasmid # 170775 ; http://n2t.net/addgene:170775 ; RRID:Addgene_170775)
  • For your References section:

    Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH. Chin MY, Patwardhan AR, Ang KH, Wang AL, Alquezar C, Welch M, Nguyen PT, Grabe M, Molofsky AV, Arkin MR, Kao AW. ACS Sens. 2021 Jun 8. doi: 10.1021/acssensors.0c02318. 10.1021/acssensors.0c02318 PubMed 34102054