Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000357 | pAAVS1-P-MCS | 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
- Vector Name:
- pAAVS1-P-MCS
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5504 bp
- Type:
- Mammalian Expression ; donor vector (human)
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
pAAVS1-P-MCS 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.
pAAVS1-P-MCS vector Sequence
LOCUS 40924_3296 5504 bp DNA circular SYN 13-MAY-2021
DEFINITION donor vector for AAVS1 targeting (puromycin selection) with multiple
cloning site.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5504)
AUTHORS Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T,
Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K
TITLE Engineering the AAVS1 locus for consistent and scalable transgene
expression in human iPSCs and their differentiated derivatives.
JOURNAL Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi:
10.1016/j.ymeth.2015.12.012.
PUBMED 26707206
REFERENCE 2 (bases 1 to 5504)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5504)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Methods.
2015 Dec 18. pii: S1046-2023(15)30181-X. doi:
10.1016/j.ymeth.2015.12.012."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5504
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 21..125
/label=AmpR promoter
CDS 126..983
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1157..1745
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1899..1916
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2033..2054
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2069..2099
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2107..2123
/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 2131..2147
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2168..2186
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 2199..3002
/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 3009..3034
/label=SA
/note="splice acceptor site"
CDS 3058..3111
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 3121..3717
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 3758..3982
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
misc_feature 4023..4843
/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(4861..4879)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4886..4902)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 5044..5499
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"