pEnt R3L2 TetO(sh) mCh-Rs1-2
(Plasmid
#24417)
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Depositing Lab
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Publication
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 24417 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepENTR2B
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Backbone manufacturerInvitrogen
- Backbone size w/o insert (bp) 2700
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Vector typeEntry Vector
Growth in Bacteria
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Bacterial Resistance(s)Kanamycin, 50 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Copy numberUnknown
Gene/Insert
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Gene/Insert nameRs1
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Alt namepEntR3L2 TetO-mCh-Rs1
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SpeciesH. sapiens (human)
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Insert Size (bp)1166
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MutationHuman 5HT4B receptor with D100A mutation, generating the RASSL Rs1. Construct expresses both mCherry and Rs1 via a P2A ribosomal skip sequence
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Tags
/ Fusion Proteins
- mCherry (N terminal on backbone)
- FLAG (N terminal on insert)
Cloning Information
- Cloning method Restriction Enzyme
- 5′ sequencing primer pENTR-F (Common Sequencing Primers)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
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
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.
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For your Materials & Methods section:
pEnt R3L2 TetO(sh) mCh-Rs1-2 was a gift from Edward Hsiao (Addgene plasmid # 24417 ; http://n2t.net/addgene:24417 ; RRID:Addgene_24417) -
For your References section:
Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice. Hsiao EC, Nguyen TD, Ng JK, Scott MJ, Chang WC, Zahed H, Conklin BR. Stem Cell Res Ther. 2011 Mar 4. 2(2):11. 10.1186/scrt52 PubMed 21375737