TFEB promoter-luciferase reporter vector (V000908)

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Price Information

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V000908	TFEB promoter-luciferase reporter	In stock, instant shipping	Buy one, get one free!

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

PGC-1α promoted htt turnover and the elimination of protein aggregates by activating transcription factor EB (TFEB), a master regulator of the autophagy-lysosome pathway.Placing PGC-1α upstream of TFEB. PGC-1α and TFEB thus hold great promise as therapies for HD and other neurodegenerative proteinopathies

Vector Name:: TFEB promoter-luciferase reporter

Antibiotic Resistance:: Ampicillin

Length:: 7001 bp

Type:: Luciferase

Replication origin:: ori

Copy Number:: Low Copy

Cloning Method:: Restriction Enzyme

5' Primer:: RVprimer3 (CTAGCAAAATAGGCTGTCCC)

3' Primer:: EBV rev

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

TFEB promoter-luciferase reporter vector Map

Copy Sequence View Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Tsunemi T, Ashe TD, Morrison BE, Soriano KR, Au J, Roque RA, Lazarowski ER, Damian VA, Masliah E, La Spada AR. PGC-1α rescues Huntington's disease proteotoxicity by preventing oxidative stress and promoting TFEB function. Sci Transl Med. 2012 Jul 11;4(142):142ra97.

TFEB promoter-luciferase reporter vector Sequence

LOCUS       40924_48932        7001 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Reporter for activity of TFEB gene promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7001)
  AUTHORS   Tsunemi T, Ashe TD, Morrison BE, Soriano KR, Au J, Roque RA, 
            Lazarowski ER, Damian VA, Masliah E, La Spada AR
  TITLE     PGC-1alpha rescues Huntington's disease proteotoxicity by preventing
            oxidative stress and promoting TFEB function.
  JOURNAL   Sci Transl Med. 2012 Jul 11;4(142):142ra97. doi: 
            10.1126/scitranslmed.3003799.
  PUBMED    22786682
REFERENCE   2  (bases 1 to 7001)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7001)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Transl
            Med."; date: "2012-07-11"; pages: "
            10.1126/scitranslmed.3003799"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    69..117
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     misc_feature    131..222
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from
                     the human alpha-2 globin gene"
     CDS             2500..4149
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    complement(4193..4314)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4562..4579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4733..5321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5495..6352)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6353..6457)
                     /label=AmpR promoter
     rep_origin      6484..6939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"