Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001291 | AAV pEF1a-DIO-FLPo-WPRE-hGHpA | 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:
- AAV pEF1a-DIO-FLPo-WPRE-hGHpA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6892 bp
- Type:
- Mammalian Expression, AAV
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- AGCTGACAGGTGGTGGCAAT
- 3' Primer:
- TCAAGCCTCAGACAGTGGTTC
AAV pEF1a-DIO-FLPo-WPRE-hGHpA 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.
AAV pEF1a-DIO-FLPo-WPRE-hGHpA vector Sequence
LOCUS 40924_180 6892 bp DNA circular SYN 13-MAY-2021
DEFINITION An AAV vector expresses FLPo in a Cre recombinase-dependent manner..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6892)
AUTHORS Zingg B, Chou XL, Zhang ZG, Mesik L, Liang F, Tao HW, Zhang LI
TITLE AAV-Mediated Anterograde Transsynaptic Tagging: Mapping
Corticocollicular Input-Defined Neural Pathways for Defense
Behaviors.
JOURNAL Neuron. 2017 Jan 4;93(1):33-47. doi: 10.1016/j.neuron.2016.11.045.
Epub 2016 Dec 15.
PUBMED 27989459
REFERENCE 2 (bases 1 to 6892)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6892)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.neuron.2016.11.045"; journalName: "Neuron"; date:
"2017-01-4- 4"; volume: "93"; issue: "1"; pages: "33-47"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6892
/mol_type="other DNA"
/organism="synthetic DNA construct"
repeat_region 13..142
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 243..1421
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
protein_bind complement(1452..1485)
/label=lox2272
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
protein_bind complement(1536..1569)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS complement(1584..2879)
/codon_start=1
/label=FLPo
/note="nuclear-targeted site-specific recombinase"
/translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
protein_bind 2889..2922
/label=lox2272
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
protein_bind 2973..3006
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 3031..3619
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
polyA_signal 3651..4127
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
repeat_region 4167..4307
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 4382..4837
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4854..4873)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4973..4995
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5033..5051)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5119..5223
/label=AmpR promoter
CDS 5224..6081
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6255..6843
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"