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Addgene

pLY70
(Plasmid #130948)

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This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 130948 Standard format: Plasmid sent in bacteria as agar stab 1 $85
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Backbone

  • Vector backbone
    pSB4A3mut
  • Backbone manufacturer
    Original version designed by: Reshma Shetty; Mutations introduced by: unknown
  • Backbone size w/o insert (bp) 3337
  • Total vector size (bp) 11667
  • Vector type
    Bacterial Expression, Synthetic Biology

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Growth instructions
    Culture in the Lennox’s Luria-Bertani (LB) medium (10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl)
  • Copy number
    Low Copy

Gene/Insert 1

  • Gene/Insert name
    dxcas9 (3.7)
  • Species
    Streptococcus pyogenes
  • Insert Size (bp)
    4170
  • Mutation
    Mutations D10A and H840A on WT cas9 for dCas9; Synonymous mutation at R63 of dcas9 (CGT>CGC); Synonymous mutation at R447 of dcas9 (CGA>CGT); Mutations A262T, R324L, S409I, E480K, E543D, M694I and E1219V for dxCas9
  • Promoter Ptet

Cloning Information for Gene/Insert 1

  • Cloning method Unknown
  • 5′ sequencing primer TGCCACCTGACGTCTAAGAA
  • 3′ sequencing primer TGCGCTGTTAATCACTTTACTTTTATCTAATCTTG
    • (Common Sequencing Primers)

Gene/Insert 2

  • Gene/Insert name
    tetR
  • Species
    E. coli
  • Insert Size (bp)
    790
  • Mutation
    Synonymous mutation at S2 of tetR (TCT>TCA)
  • Promoter Ptet

Cloning Information for Gene/Insert 2

  • Cloning method Unknown
  • 5′ sequencing primer GAGCTGTCCGTTTGAGGCGAGTCGCTTCCGCTG
  • 3′ sequencing primer CGTCCGGTAACGCGTCCGTGGC
    • (Common Sequencing Primers)

Gene/Insert 3

  • Gene/Insert name
    rhaS
  • Species
    E. coli
  • Insert Size (bp)
    1016
  • Promoter Pcon

Cloning Information for Gene/Insert 3

  • Cloning method Ligation Independent Cloning
  • 5′ sequencing primer AGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTG
  • 3′ sequencing primer CACGAATATTTCCCGGCCAACGATAATTCAGC
    • (Common Sequencing Primers)

Gene/Insert 4

  • Gene/Insert name
    pspFΔHTH::λN22plus
  • Species
    Synthetic
  • Insert Size (bp)
    1244
  • Mutation
    The HTH domain of the pspF gene was deleted (297-326); Synonymous mutation at N86 (AAT>AAC) of the MCP domain of pspFΔHTH::MCP
  • Promoter PrhaB
  • Tag / Fusion Protein
    • pspFΔHTH::λN22plus (C terminal on insert)

Cloning Information for Gene/Insert 4

  • Cloning method Restriction Enzyme
  • 5′ cloning site XbaI (destroyed during cloning)
  • 3′ cloning site SpeI (destroyed during cloning)
  • 5′ sequencing primer ACGCTGTGGTTAACGCGCCAGTGAG
  • 3′ sequencing primer CTCGCCACGCACGGAAAACTTATGACCGTTGAC
    • (Common Sequencing Primers)

Gene/Insert 5

  • Gene/Insert name
    sfgfp
  • Species
    Synthetic
  • Insert Size (bp)
    1110
  • Promoter PpspA-LEA3B3
  • Tag / Fusion Protein
    • ASV tag (C terminal on insert)

Cloning Information for Gene/Insert 5

  • Cloning method Restriction Enzyme
  • 5′ cloning site XbaI (destroyed during cloning)
  • 3′ cloning site PstI (not destroyed)
  • 5′ sequencing primer CTGCAACTCAGTTTGCAAATGAATGCAC
  • 3′ sequencing primer ATTACCGCCTTTGAGTGAGC
    • (Common Sequencing Primers)

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

The Top10 bacterial strain should be used for downstream applications

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:

    pLY70 was a gift from Baojun Wang (Addgene plasmid # 130948 ; http://n2t.net/addgene:130948 ; RRID:Addgene_130948)

  • For your References section:

    Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria. Liu Y, Wan X, Wang B. Nat Commun. 2019 Aug 26;10(1):3693. doi: 10.1038/s41467-019-11479-0. 10.1038/s41467-019-11479-0 PubMed 31451697
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