Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010070 | pcDNA3/Flag-METTL3 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pcDNA3/Flag-METTL3
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7052 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Promoter:
- SP6
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- 5' TAATACGACTCACTATAGGG 3'
- 3' Primer:
- 5' ATTTAGGTGACACTATAG 3'
pcDNA3/Flag-METTL3 vector 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.
pcDNA3/Flag-METTL3 vector Sequence
LOCUS V010070 7052 bp DNA circular SYN 21-DEC-2021
DEFINITION Exported.
ACCESSION V010070
VERSION V010070
KEYWORDS pcDNA3/Flag-METTL3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 7052)
AUTHORS Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng
X, Dai Q, Chen W, He C
TITLE A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine
methylation.
JOURNAL Nat Chem Biol. 2014 Feb;10(2):93-5. doi: 10.1038/nchembio.1432. Epub
2013 Dec 6.
PUBMED 24316715
REFERENCE 2 (bases 1 to 7052)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7052)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nchembio.1432"; journalName: "Nat Chem Biol"; date:
"2014-02"; volume: "10"; issue: "2"; pages: "93-5"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7052
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 23..44
/label="F1ori-F"
/note="F1 origin, forward primer"
primer_bind complement(163..183)
/label="pBABE 3'"
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 207..417
/label="SV40 promoter"
/note="SV40 early promoter"
CDS 492..1283
/label="NeoR/KanR"
/note="aminoglycoside phosphotransferase"
polyA_signal 1460..1593
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(1630..1646)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(1654..1670)
/label="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(1678..1708)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(1723..1744)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1861..1878)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(2032..2620)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2794..3651)
/label="AmpR"
/note="beta-lactamase"
promoter complement(3652..3756)
/label="AmpR promoter"
primer_bind complement(3831..3850)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 4022..4401
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 4402..4605
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 4650..4668
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 4685..4708
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 4709..6445
/gene="METTL3"
/label="N6-adenosine-methyltransferase catalytic subunit"
/note="N6-adenosine-methyltransferase catalytic subunit
from Homo sapiens. Accession#: Q86U44"
promoter complement(6464..6482)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
polyA_signal 6508..6732
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 6778..7052
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"