Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V002058 | px330_DBH-p2a-FLPo | 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:
- px330_DBH-p2a-FLPo
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8509 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Sun JJ, Ray R.
- Promoter:
- CBh
px330_DBH-p2a-FLPo vector 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.
px330_DBH-p2a-FLPo vector Sequence
LOCUS 40924_47008 8509 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector px330_DBH-p2a-FLPo, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8509)
AUTHORS Sun JJ, Ray R.
TITLE Generation of Two Noradrenergic-Specific
Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using
CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells
JOURNAL PLoS ONE 11 (7), E0159474 (2016)
PUBMED 27441631
REFERENCE 2 (bases 1 to 8509)
AUTHORS Sun JJ, Ray R.
TITLE Direct Submission
JOURNAL Submitted (27-APR-2016) Neuroscience, Baylor College of Medicine,
One Baylor Plaza Room T707, Houston, TX 77030, USA
REFERENCE 3 (bases 1 to 8509)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8509)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE";
date: "2016"; volume: "11"; issue: "7"; pages: "E0159474"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(27-APR-2016) Neuroscience, Baylor College of Medicine, One Baylor
Plaza Room T707, Houston, TX 77030, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8509
/mol_type="other DNA"
/organism="synthetic DNA construct"
regulatory 1..270
/label=CAG
/note="CAG"
/regulatory_class="enhancer"
promoter 284..561
/label=chicken beta-actin promoter
intron 562..790
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 802..811
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 811..876
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 883..903
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 928..5028
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 5029..5076
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5110..5317
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 5326..5466
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5541..5996
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6278..6382
/label=AmpR promoter
CDS 6383..7240
/label=AmpR
/note="beta-lactamase"
rep_origin 7414..8002
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 8064..8304
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 8314..8333
/label=DBH-p2a-FLPo sgRNA
/note="DBH-p2a-FLPo sgRNA"
misc_RNA 8334..8409
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 8506..8509
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"