pLY164
(Plasmid #184140)

• Purpose: Reporter circuit for hijacking RFP mRNA as CRISPR RNA (target site 3)
• Depositing Lab: Baojun Wang
• Publication: Liu et al Nat Commun. 2022 Apr 11;13(1):1937. doi: 10.1038/s41467-022-29604-x.

Backbone

• Vector backbone: pSB4A3mut
• Backbone size w/o insert (bp): 3337
• Total vector size (bp): 11648
• Modifications to backbone: Original version designed by: Reshma Shetty; Mutations introduced by: unknown
• 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: dCas9
• Species: Streptococcus pyogenes
• Insert Size (bp): 4170
• Mutation: Mutations D10A and H840A on WT cas9. Synonymous mutation at R63 of dcas9 (CGT>CGC); Synonymous mutation at R447 of dcas9 (CGA>CGT)
• Promoter: Ptet

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: TGCCACCTGACGTCTAAGAA
• 3′ sequencing primer: TGCGCTGTTAATCACTTTACTTTTATCTAATCTTG

Gene/Insert 2

• Gene/Insert name: tetR
• Species: Escherichia coli
• Insert Size (bp): 789
• 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

Gene/Insert 3

• Gene/Insert name: pspFΔHTH::λN22plus
• Species: Synthetic
• Insert Size (bp): 1244
• Mutation: The HTH domain of the pspF gene was deleted (297-326). Mutations D2N and Q4R in the λN22 domain.
• Promoter: PrhaB
• Tag / Fusion Protein:
  • λN22plus (C terminal on insert)

Cloning Information for Gene/Insert 3

• 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

Gene/Insert 4

• Gene/Insert name: sfgfp
• Species: Synthetic
• Insert Size (bp): 1110
• Promoter: PpspA-R3
• Tag / Fusion Protein:
  • ASV tag (C terminal on insert)

Cloning Information for Gene/Insert 4

• 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

Gene/Insert 5

• Gene/Insert name: rhaS
• Species: Escherichia coli
• Insert Size (bp): 1016
• Promoter: Pcon

Cloning Information for Gene/Insert 5

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: AGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTG
• 3′ sequencing primer: CACGAATATTTCCCGGCCAACGATAATTCAGC

Terms and Licenses

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