Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005932 | pT3-myr-AKT-HA | 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:
- pT3-myr-AKT-HA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7091 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Promoter:
- EF-1α
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- EF-1a-Fwd
- 3' Primer:
- pT3EF1a-R 5'-TGACACCTACTCAGACAATG
pT3-myr-AKT-HA 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.
pT3-myr-AKT-HA vector Sequence
LOCUS 40924_42264 7091 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 7091)
AUTHORS Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G,
Delogu S, Zimmermann A, Ericsson J, Brozzetti S, Staniscia T, Chen
X, Dombrowski F, Evert M
TITLE Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling,
promotes development of human hepatocellular carcinoma.
JOURNAL Gastroenterology. 2011 Mar;140(3):1071-83. Epub 2010 Dec 11.
PUBMED 21147110
REFERENCE 2 (bases 1 to 7091)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7091)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Gastroenterology."; date: "2011-03"; volume: "140(3)"; pages:
"1071-83. Epub 2010 Dec 11"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7091
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..1179
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
promoter 1196..1214
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1293..1317
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 1338..1358
/codon_start=1
/label=myr
/note="N-myristoylation signal from Src kinase (Pellman et
al., 1985; Kaplan et al., 1988)"
/translation="MGSSKSK"
CDS 1371..2810
/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 2811..2837
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
protein_bind complement(2857..2881)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS 2934..2975
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
polyA_signal 3019..3243
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 3313..3346
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
primer_bind 3402..3422
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
primer_bind complement(3804..3820)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4029..4048)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4148..4170
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4208..4226)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4294..4398
/label=AmpR promoter
CDS 4399..5256
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5430..6018
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6172..6189
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6306..6327
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6342..6372
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6380..6396
/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 6404..6420
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6846..6865)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
protein_bind complement(6918..6951)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 7091
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"