tetO-ALN
(Plasmid #43918)

• Depositing Lab: John Gearhart
• Publication: Addis et al PLoS One. 2011;6(12):e28719. doi: 10.1371/journal.pone.0028719. Epub 2011 Dec 9.

Backbone

• Vector backbone: FU-tetO-Gateway
• Backbone manufacturer: Addgene plasmid 43914
• Backbone size w/o insert (bp): 8600
• Total vector size (bp): 12400
• Vector type: Mammalian Expression, Lentiviral ; Doxycycline inducible
• Selectable markers: Zeo marker is outside the LTRs and will not be packaged into virus.

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use recA cells, such as STBL3 (Invitrogen)
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ASCL1-2A-LMX1B-2A-NURR1
• Alt name: ASCL1
• Alt name: LMX1B
• Alt name: NURR1 (NR4A2)
• Species: H. sapiens (human)
• Insert Size (bp): 3800
• Mutation: Gylcine-serine-glycine (GSG) linker followed by viral 2A peptide sequence inserted between each ORF
• GenBank ID: DQ894571/EL736297/EU832740 (Genbank Accession Numbers) 100009031/13957/100067769 (Open Biosystems IDs)
• Entrez Gene: ASCL1 (a.k.a. ASH1, HASH1, MASH1, bHLHa46)
• Entrez Gene: LMX1B (a.k.a. FSGS10, LMX1.2, NPS1)
• Entrez Gene: NR4A2 (a.k.a. HZF-3, IDLDP, NOT, NURR1, RNR1, TINUR)
• Promoter: TRE promoter, Tet-ON
• Tags / Fusion Proteins:
  • V5 (C terminal on backbone)
  • 6xHis (C terminal on backbone)

Cloning Information

• Cloning method: Gateway Cloning
• 5′ sequencing primer: LNCX; T7
• 3′ sequencing primer: WPRE-R

Resource Information

• Supplemental Documents:
  • tetO-ALN annotated genbank file
• A portion of this plasmid was derived from a plasmid made by: ASCL1, LMX1B, and NURR1 ORF cDNAs purchased from Open Biosystems (Now part of Thermo Scientific, Waltham, MA)
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

To make a drug-inducible lentiviral vector that is compatible with Gateway recombination (Invitrogen), the depositing laboratory removed the OCT4 open reading frame from FU-tetO-hOCT4 (Addgene plasmid 19778) and replaced it with a Gateway cassette cloned from pEF-DEST51 (Invitrogen) to generate FU-tetO-Gateway (Addgene plasmid #43914).

Open reading frames of human ASCL1, LMX1B, and NURR1 were purchased from Open Biosystems and amplified using the following PCR primers:

B1-ASCL1-FWD: GGGGacaagtttgtacaaaaaagcaggctACCATGGAAAG​CTCTGCCAAG;
ASCL1-REV: CATCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCACC​GGATCCGAACCAGTTGGTGAAGTC;
LMX1B-FWD: CTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCC​GGGCCTATGTTGGACGGCATCAAG;
LMX1B-REV: ACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCGG​ATCCGGAGGCGAAGTAGGAACT;
NURR1-FWD: GAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGG​CCCTATGCCTTGTGTTCAGGCG;
B2-NURR1-REV: GGGGaccactttgtacaagaaagctgggt[A]GAAAGGTAAAGTGTCCAG
(extra [A] added to maintain reading frame with C-terminal V5-tag).

The 3 ORFs were then joined via recombinant PCR such that viral 2A peptide sequences separate the three genes, using B1-ASCL1-FWD and B2-NURR1-REV. A glycine-serine-glycine (GSG) linker was included upstream of each 2A sequence to facilitate protein cleavage.

The ASCL1-P2A-LMX1B-T2A-NURR1(ALN) cassette was cloned into pDONR221, and subsequently cloned into FU-tetO-Gateway via Gateway recombination to produce the tetO-ALN vector.

Cleavage at 2A sites was verified by performing in vitro transcription and translation using the TnT T7 Coupled Reticulocyte Lysate System (Promega L4610) in the presence of biotinylated lysine (Promega L5061). 5 µL of each TnT-generated protein sample was resolved via PAGE, transferred to PVDF membrane, incubated with streptavidin-conjugated horseradish peroxidase (Cell Signaling Technology 3999, 1:2000), and detected with ECL Plus (GE Healthcare RPN2132).