Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007069 | secreted BirA-Flag | 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:
- secreted BirA-Flag
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7001 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CMV forward
secreted BirA-Flag 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.
secreted BirA-Flag vector Sequence
LOCUS 40924_48778 7001 bp DNA circular SYN 13-MAY-2021
DEFINITION Flag-tagged biotinylation enzyme for co-expression with
bio-proteins.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7001)
AUTHORS Bushell KM, Sollner C, Schuster-Boeckler B, Bateman A, Wright GJ
TITLE Large-scale screening for novel low-affinity extracellular protein
interactions.
JOURNAL Genome Res. 2008 Apr;18(4):622-30. Epub 2008 Feb 22.
PUBMED 18296487
REFERENCE 2 (bases 1 to 7001)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7001)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Genome
Res."; date: "2008-04"; volume: "18(4)"; pages: "622-30. Epub 2008
Feb 22"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7001
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 154..533
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 534..737
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 734..758
/label=LNCX
/note="Human CMV promoter, forward primer"
primer_bind 1239..1258
/label=pMT2-F
/note="Synthetic intron, forward primer"
CDS 1362..2321
/codon_start=1
/label=BirA
/note="E. coli biotin protein ligase"
/translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
CDS 2328..2351
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
polyA_signal 2381..2436
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(2435..2454)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(4672..4690)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4758..4862
/label=AmpR promoter
CDS 4863..5720
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5894..6482
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6636..6653
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6770..6791
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6806..6836
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6844..6860
/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 6868..6884
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"