pEnt R3L2 TetO(sh) mCh-Rs1-2
(Plasmid #24417)

• Purpose: Human 5HT4B receptor with D100A mutation, generating the RASSL Rs1. Construct expresses both mCherry and Rs1 via a P2A ribosomal skip sequence. Plasmid also known as pEntR3L2 TetO-mCh-Rs1.
• Depositing Lab: Edward Hsiao
• Publication: Hsiao et al Stem Cell Res Ther. 2011 Mar 4. 2(2):11.

Backbone

• Vector backbone: pENTR2B
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 2700
• Vector type: Entry Vector

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Rs1
• Species: H. sapiens (human)
• Insert Size (bp): 1166
• Mutation: D100A
• Promoter: short TetO, miniCMV
• Tags / Fusion Proteins:
  • mCherry-P2A (N terminal on backbone)
  • FLAG (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XhoI (not destroyed)
• 3′ cloning site: AflII (not destroyed)
• 5′ sequencing primer: pENTR-F

Terms and Licenses

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

Depositor Comments

Contains Gateway R3L2 sites. TetO is missing 2 repeats. Drives expression of mCherry-P2A-Rs1 cassette. Contains insulator sequence.

Plasmid was constructed as follows: The pEntr2B entry vector was digested with AflII and XhoI to remove the attL1 and ccdb genes. Oligos containing the attR3 site flanked by AflII on the 5' end and a polylinker (NotI-PacI-Bsu36I-NdeI-XhoI) on the 3' end were ligated to generate the empty pEntR3L2-MCS intermediate. Using PCR cloning, the TetO-beta globin intron sequence from pTetO (pUHD10.3) was cloned into the NotI/PacI sites in the reverse orientation, and a 3' SbfI site was introduced. The pA sequence from pDest27 was then cloned into the NotI/SbfI sites, again in reverse orientation. A LoxP site was introduced into the NdeI/XhoI sites, allowing full excision of the targeted construct when used together with the LoxP site in the pEntl1L3 vector. The CBG core insulator was then cloned into the Bsu36I/NdeI sites to generate the starting entry vector pEntR3L2 TetO(fl)-2 (Addgene plasmid number 24416).
The mCherry-P2A-Rs1 RASSL cassette was cloned into the NotI site to generate pEntR3L2 TetO(sh) mCh-Rs1-2. The Rs1 was PCR amplified from pUNIV-SIG-5HTR4D100A and the mCherry was PCR cloned from pRset-mCherry. The self-cleaving 2A site generates two separate peptides in equal concentrations via a ribosomal “skip” mechanism just before the C-terminal end of the 2A peptide and has been useful for making multicistronic reporters. During sequencing of the final pEntR3L2 TetO(sh) mCh-Rs1-2 construct (abbreviated pEntR3L2 TetO-mCh-Rs1), the TetO region was noted to carry a deletion of two of the 7 tTA binding site repeats. However, the shortened TetO retained comparable function to the full-length TetO (designated with “fl” for full length) in separate in vitro assays.