Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010075 | pDestTol2pA2-U6:gRNA | 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:
- pDestTol2pA2-U6:gRNA
- Antibiotic Resistance:
- Chloramphenicol, Ampicillin
- Length:
- 6352 bp
- Type:
- CRISPR ; Tol2 destination vector for Gateway
- Replication origin:
- ori
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- GAGGATCATAATCAGCCATA
pDestTol2pA2-U6:gRNA 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.
pDestTol2pA2-U6:gRNA vector Sequence
LOCUS 40924_14495 6352 bp DNA circular SYN 13-MAY-2021
DEFINITION Destination vector for Gateway based on vector #394 from the
Tol2Kit, containing a gRNA scaffold under the control of a zebrafish
U6 promoter..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6352)
AUTHORS Ablain J, Durand EM, Yang S, Zhou Y, Zon LI
TITLE A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in
Zebrafish.
JOURNAL Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi:
10.1016/j.devcel.2015.01.032.
PUBMED 25752963
REFERENCE 2 (bases 1 to 6352)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6352)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Dev Cell.
2015 Mar 4. pii: S1534-5807(15)00075-1. doi:
10.1016/j.devcel.2015.01.032."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6352
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 20..124
/label=AmpR promoter
CDS 125..982
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1156..1744
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1898..1915
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2032..2053
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2068..2098
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2106..2122
/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 2130..2146
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2167..2185
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 2222..2238
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
polyA_signal 2910..3044
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
misc_RNA 3443..3518
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind 3531..3547
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3555..3678
/label=attR3
/note="recombination site for the Gateway(R) LR reaction"
promoter 3703..3733
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 3787..4464
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
DPAY"
CDS 4787..5089
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(5133..5257)
/label=attR4
/note="recombination site for the Gateway(R) LR reaction"
primer_bind complement(5276..5292)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6188..6209
/label=F1ori-F
/note="F1 origin, forward primer"