Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006735 | AAVS1-Neo-M2rtTA | 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
AAVS1 donor vector for genomic targeting
- Vector Name:
- AAVS1-Neo-M2rtTA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10080 bp
- Type:
- Mammalian Expression Vectors
- Replication origin:
- ori
- Selection Marker:
- Neomycin (select with G418)
- Promoter:
- CAG
AAVS1-Neo-M2rtTA 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.
AAVS1-Neo-M2rtTA vector Sequence
LOCUS 40924_170 10080 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10080)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 10080)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10080
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 33..836
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
misc_feature 843..868
/label=SA
/note="splice acceptor site"
CDS 892..945
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 961..1761
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase from Tn5"
polyA_signal 1836..2291
/label=PGK poly(A) signal
/note="mouse phosphoglycerate kinase 1 polyadenylation
signal"
enhancer 2389..2692
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2694..2971
/note="chicken beta-actin promoter"
intron 2973..3990
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
CDS 4090..4833
/label=rtTA-Advanced
/note="improved tetracycline-controlled transactivator"
polyA_signal complement(4846..4980)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
misc_feature 5310..6146
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter complement(6187..6205)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6212..6228)
/label=M13 fwd
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
CDS 6366..6665
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
CDS complement(8068..8925)
/label=AmpR
/note="beta-lactamase"
rep_origin 9049..9637
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 9925..9946
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9961..9991
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9999..10015
/label=lac repressor encoded by lacI binding site
/bound_moiety="lac repressor encoded by lacI"
/note="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 10023..10039
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 10060..10078
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"