Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V016629 | pRW006-SM-destination-pro35S-Cas9 | 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:
- pRW006-SM-destination-pro35S-Cas9
- Antibiotic Resistance:
- Streptomycin
- Length:
- 11115 bp
- Type:
- Gene knockout
- Replication origin:
- pSa ori
- Host:
- Plants
- Promoter:
- CaMV35S(short)
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 30℃
pRW006-SM-destination-pro35S-Cas9 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.
pRW006-SM-destination-pro35S-Cas9 vector Sequence
LOCUS 62056_19115 11115 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11115)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11115
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 534..556
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA
(truncated)"
promoter 1086..1431
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 1460..1507
/codon_start=1
/label=2xFLAG
/note="two tandem FLAG(R) epitope tags"
/translation="DYKDDDDKDYKDDDDK"
CDS 1514..1534
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
intron 2996..3184
/label=IV2 intron
/note="modified second intron of the potato ST-LS1 gene
(Vancanneyt et al., 1990)"
CDS 3350..5848
/codon_start=1
/label=Cas9(C)
/note="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
IHQSITGLYETRIDLSQLGGD"
CDS 5849..5896
/codon_start=1
/label=nucleoplasmin NLS
/note="bipartite nuclear localization signal from
nucleoplasmin"
/translation="KRPAATKKAGQAKKKK"
terminator 5902..6543
/label=E9 terminator
/note="terminator and polyadenylation signal from the pea
rbcS-E9 gene"
promoter complement(7133..7163)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(7178..7199)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
terminator complement(7712..7964)
/label=MAS terminator
/note="mannopine synthase terminator"
CDS complement(7973..8659)
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="DNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLK
VTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVT
VTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRL
KLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTG
GMDELYK"
misc_feature 9134..9158
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
rep_origin complement(9249..9837)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"