Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001239 | pFRT/TO/HIS/FLAG/HA-ALKBH5 | 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:
- pFRT/TO/HIS/FLAG/HA-ALKBH5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6875 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- CMV
- 3' Primer:
- BGH
pFRT/TO/HIS/FLAG/HA-ALKBH5 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.
pFRT/TO/HIS/FLAG/HA-ALKBH5 vector Sequence
LOCUS 40924_20501 6875 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 6875)
AUTHORS Baltz AG, Munschauer M, Schwanhausser B, Vasile A, Murakawa Y,
Schueler M, Youngs N, Penfold-Brown D, Drew K, Milek M, Wyler E,
Bonneau R, Selbach M, Dieterich C, Landthaler M
TITLE The mRNA-Bound Proteome and Its Global Occupancy Profile on
Protein-Coding Transcripts.
JOURNAL Mol Cell. 2012 Jun 8;46(5):674-90.
PUBMED 22681889
REFERENCE 2 (bases 1 to 6875)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6875)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell.";
date: "2012-06-8"; volume: "46(5)"; pages: "674-90"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6875
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 67..108
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
polyA_signal 152..376
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(509..528)
/label=F1ori-R
/note="F1 origin, reverse primer"
protein_bind 660..707
/label=FRT
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS 715..1734
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
"
polyA_signal 1867..2000
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2037..2053)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2061..2077)
/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(2085..2115)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(2130..2151)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(2268..2285)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2439..3027)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3201..4058)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4059..4163)
/label=AmpR promoter
primer_bind complement(4238..4257)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 4429..4808
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4809..5012
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 5014..5032
/label=tet operator
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 5035..5053
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 5063..5087
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 5187..5210
/codon_start=1
/label=8xHis
/note="8xHis affinity tag"
/translation="HHHHHHHH"
CDS 5214..5237
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
CDS 5238..5264
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
protein_bind 5280..5304
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
protein_bind complement(6690..6714)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS 6767..6808
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"