Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012090 | pBSFI-myr-akt-T308A/S473A | 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:
- pBSFI-myr-akt-T308A/S473A
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4474 bp
- Type:
- Adaptor Plasmid
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T3
- 3' Primer:
- T7
pBSFI-myr-akt-T308A/S473A 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.
pBSFI-myr-akt-T308A/S473A vector Sequence
LOCUS 40924_7406 4474 bp DNA circular SYN 13-MAY-2021
DEFINITION cDNA of Akt1 with myristyolation signal and point mutation from T to
A at position 308 and S to A at position 473 in cloning vector.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4474)
AUTHORS Aoki M, Batista O, Bellacosa A, Tsichlis P, Vogt PK
TITLE The akt kinase: molecular determinants of oncogenicity.
JOURNAL Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14950-5.
PUBMED 9843996
REFERENCE 2 (bases 1 to 4474)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4474)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl
Acad Sci U S A."; date: "1998-12-8"; volume: "95(25)"; pages:
"14950-5"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4474
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 63..83
/codon_start=1
/label=myr
/note="N-myristoylation signal from Src kinase (Pellman et
al., 1985; Kaplan et al., 1988)"
/translation="MGSSKSK"
CDS 96..1535
/codon_start=1
/label=Akt1
/note="human serine/threonine protein kinase activated by
growth factors"
/translation="MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
DQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQ
TVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
LDKDGHIKITDFGLCKEGIKDGATMKAFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDA
KEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
ECVDSERRPHFPQFAYSASGTA"
CDS 1536..1562
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
promoter complement(1632..1650)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1657..1673)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin complement(1814..2269)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2296..2400
/label=AmpR promoter
CDS 2401..3258
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3432..4020
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4174..4191
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4308..4329
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4344..4374
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4382..4398
/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)."
primer_bind 4406..4422
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 4443..4461
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"