Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011885 | pX601-mCherry | 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:
- pX601-mCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8122 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Promoter:
- U6
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- Cas9-HidIII-S: gccccgtcgtgaagagaagcttcatcc
- 3' Primer:
- mCherry –EcoR1-AS:cgtagaattcttacttgtacagctcgtccatgccgccggt
pX601-mCherry 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.
pX601-mCherry vector Sequence
LOCUS 40924_47073 8122 bp DNA circular SYN 13-MAY-2021
DEFINITION Staphylococcus aureus (SaCas9) conjugated with mCherry.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8122)
AUTHORS Ye L, Wang J, Tan Y, Beyer AI, Xie F, Muench MO, Kan YW
TITLE Genome editing using CRISPR-Cas9 to create the HPFH genotype in
HSPCs: An approach for treating sickle cell disease and
beta-thalassemia.
JOURNAL Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10661-5. doi:
10.1073/pnas.1612075113. Epub 2016 Sep 6.
PUBMED 27601644
REFERENCE 2 (bases 1 to 8122)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8122)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1612075113"; journalName: "Proc Natl Acad Sci U S A";
date: "2016-09-20- 20"; volume: "113"; issue: "38"; pages: "10661-5"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8122
/mol_type="other DNA"
/organism="synthetic DNA construct"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
enhancer 154..533
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 534..737
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 756..765
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 768..788
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 813..3968
/codon_start=1
/label=SaCas9
/note="Cas9 endonuclease from the Staphylococcus aureus
Type II CRISPR/Cas system"
/translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
IKKG"
CDS 3969..4016
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 4017..4070
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 4071..4778
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
polyA_signal 4812..5019
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 5027..5267
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 5296..5371
/label=Sa gRNA scaffold
/note="guide RNA scaffold for the Staphylococcus aureus
CRISPR/Cas9 system"
repeat_region 5385..5525
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5600..6055
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6072..6091)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6191..6213
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(6251..6269)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6337..6441
/label=AmpR promoter
CDS 6442..7299
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7473..8061
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"