Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017502 | eeBxb1 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The eeBxb1 designed for expressing evolved and engineered Bxb1 in the mammalian cells
- Vector Name:
- eeBxb1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5801 bp
- Type:
- Mammalian Expression Vectors
- Promoter:
- CMV
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
eeBxb1 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.
References
- Pandey S, Gao XD, Krasnow NA, et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat Biomed Eng. Published online June 10, 2024. doi:10.1038/s41551-024-01227-1
eeBxb1 vector Sequence
LOCUS Exported 5801 bp DNA circular SYN 06-SEP-2024
DEFINITION Plasmid for expressing evolved and engineered Bxb1 in the mammalian
cells.
ACCESSION .
VERSION .
KEYWORDS eeBxb1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5801)
AUTHORS Pandey S, Gao XD, Krasnow NA, McElroy A, Tao YA, Duby JE, Steinbeck
BJ, McCreary J, Pierce SE, Tolar J, Meissner TB, Chaikof EL, Osborn
MJ, Liu DR
TITLE Efficient site-specific integration of large genes in mammalian
cells via continuously evolved recombinases and prime editing.
JOURNAL Nat Biomed Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1.
PUBMED 38858586
REFERENCE 2 (bases 1 to 5801)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Biomed
Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1."
FEATURES Location/Qualifiers
source 1..5801
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 1..589
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 490..509
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 743..760
/label=L4440
/note="L4440 vector, forward primer"
promoter complement(834..938)
/gene="bla"
/label=AmpR promoter
primer_bind 1006..1024
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 1051..1081
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind 1089..1105
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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 1094..1116
/label=M13/pUC Reverse
/note="In lacZ gene"
enhancer 1135..1438
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1439..1642
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 1593..1613
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 1639..1663
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 1758..1776
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1758..1775
/label=SP6
/note="SP6 promoter, forward primer"
CDS 1814..3316
/codon_start=1
/transl_table=11
/gene="Bxb1"
/product="Bxb1 integrase"
/label=Bxb1 integrase
/note="Bxb1 large serine integrase from Mycobacterium
bacteriophage Bxb1 (Ghosh et al. 2003)."
/protein_id="AFF61396.1"
/translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
VSGAVDPFDRKRRPNLARWLAFEEQPFDAIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
REPQGRKWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAIELAEVNAELVDLTSLIGSPAYRAG
SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
polyA_signal 3728..3862
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3765..3784)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3819..3838
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 3988..4442
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4075..4094)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4284..4305
/label=F1ori-F
/note="F1 origin, forward primer"
CDS 4729..5589
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(4947..4966)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
protein_bind complement(5691..5724)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."