4xCSL-luciferase vector (V006516)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V006516	4xCSL-luciferase	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

The 4xCSL - luciferase plasmid is an important tool in Notch signaling research. It is constructed from the CBF1/pGL2 - GLO TATA CAT plasmid. A fragment containing multimerized high affinity CSL sites (4 CGTGGGAA) is inserted into a BglII/BamHI - digested RSV - TATA pGL2 vector, which has a TATA box from Rous sarcoma virus to reduce basal luciferase activity.
This plasmid is widely used in transfection assays. In experiments, it is often transfected into cells along with Notch constructs and other control plasmids. The luciferase activity of the transfected cells is then measured to assess the activation of CSL - dependent pathways. For example, in studies on murine Notch homologs (N1 - 4), it was used to determine whether truncated Notch proteins could activate a CSL - dependent reporter in the absence of presenilin proteins.
The 4xCSL - luciferase plasmid offers several advantages. It provides a specific and sensitive way to study Notch signaling and its regulation of target genes. The presence of multiple CSL sites enhances the detection of CSL - dependent transcriptional activation, allowing for more accurate analysis of the signaling pathway.

Vector Name:: 4xCSL-luciferase

Antibiotic Resistance:: Ampicillin

Length:: 5715 bp

Type:: Mammalian Expression, Luciferase

Replication origin:: ori

Copy Number:: High Copy

Cloning Method:: Restriction Enzyme

5' Primer:: F1ori-F (GTGGACTCTTGTTCCAAACTGG)

3' Primer:: LucNRev

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

4xCSL-luciferase 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

Saxena MT, Schroeter EH, Mumm JS, Kopan R. Murine notch homologs (N1-4) undergo presenilin-dependent proteolysis. J Biol Chem. 2001 Oct 26;276(43):40268-73. doi: 10.1074/jbc.M107234200. Epub 2001 Aug 22. PMID: 11518718.

4xCSL-luciferase vector Sequence

LOCUS       40924_90        5715 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5715)
  AUTHORS   Saxena MT, Schroeter EH, Mumm JS, Kopan R
  TITLE     Murine notch homologs (N1-4) undergo presenilin-dependent 
            proteolysis.
  JOURNAL   J Biol Chem. 2001 Oct 26;276(43):40268-73. Epub 2001 Aug 22.
  PUBMED    11518718
REFERENCE   2  (bases 1 to 5715)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5715)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol
            Chem."; date: "2001-10-26"; volume: "276(43)"; pages: "40268-73.
            Epub 2001 Aug 22"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    86..207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             425..2074
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     intron          2318..2383
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2513..2533
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    2958..3092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3234..3251)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3405..3993)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4167..5024)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5025..5129)
                     /label=AmpR promoter
     rep_origin      5156..5611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"