pNFkB-Luc vector (Cat. No.: V004337)

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Basic Information

Note: NFkB reporter vector is utilized for detecting NFkB (Nuclear factor of kappa light polypeptide gene) transactivation in a cell-based assay.

Name:: pNFkB-Luc

Antibiotic Resistance:: Ampicillin

Length:: 5711 bp

Type:: Reporter vector

Replication origin:: ori

Source/Author:: Zheng C-F.

Selection Marker:: Fluc

Promoter:: minP

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

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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.

pNFkB-Luc vector (Cat. No.: V004337) Sequence

LOCUS       pNFkB-Luc               5711 bp    DNA     circular SYN 26-DEC-2025
DEFINITION  Reporter vector pNFkB-Luc, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5711)
  AUTHORS   Zheng C-F.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-MAR-1998) Technical Services, Stratagene, 11011 N. 
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   2  (bases 1 to 5711)
  AUTHORS   Zheng C-F.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-JAN-1999) Technical Services, Stratagene, 11011 N. 
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 5711)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5711)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted
            (11-MAR-1998) Technical Services, Stratagene, 11011 N. Torrey Pines
            Rd., La Jolla, CA 92037, USA"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (20-JAN-1999) Technical Services, Stratagene, 11011 N. Torrey Pines
            Rd., La Jolla, CA 92037, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /label=AmpR promoter
     CDS             137..994
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(1942..2082)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     polyA_signal    2321..2455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2549..2683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    2692..2758
                     /label=NFkB response elements
     promoter        2769..2800
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             2827..4476
                     /label=luciferase
                     /note="firefly luciferase"
     intron          4610..4675
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4872..5227
                     /codon_start=1
                     /label=SV40 T-ANTIGEN
                     /translation="MLCLVIELLLALLFTPQRKKLHCYTRKLWKNIL*PL*VGITVIII
                     TYCFFLLHTGIECLLLITMLKNCVPLAF*FVKGLIRNI*CIVP*LEIIISHTTFVEVLL
                     ALKNLPHLPLNLKH"
     polyA_signal    5250..5384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"