Price Information
Cat No. | Plasmid Name | Availability | Add to cart |
---|---|---|---|
V017390 | pT3EF1aH-myr-Akt | 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:
- pT3EF1aH-myr-Akt
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6694 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Promoter:
- EF-1α
- Growth Temperature:
- 37℃
pT3EF1aH-myr-Akt 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.
pT3EF1aH-myr-Akt vector Sequence
LOCUS 62056_20530 6694 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6694) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..6694 /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin complement(206..794) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(968..1825) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(1826..1930) /label=AmpR promoter primer_bind 2404..2420 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 2944..4122 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" protein_bind 4153..4177 /label=attB1 /note="recombination site for the Gateway(R) BP reaction" CDS 4212..4232 /codon_start=1 /label=myr /note="N-myristoylation signal from Src kinase (Pellman et al., 1985; Kaplan et al., 1988)" /translation="MGSSKSK" CDS 4245..5684 /codon_start=1 /label=Akt1 /note="human serine/threonine protein kinase activated by growth factors" /translation="MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV DQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQ TVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM LDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDA KEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM ECVDSERRPHFPQFSYSASGTA" CDS 5685..5711 /codon_start=1 /label=HA /note="HA (human influenza hemagglutinin) epitope tag" /translation="YPYDVPDYA" protein_bind complement(5744..5768) /label=attB2 /note="recombination site for the Gateway(R) BP reaction" polyA_signal 5856..6080 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6498..6514) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(6522..6538) /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(6546..6576) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(6591..6612) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP."