Basic Vector Information
- Vector Name:
- G1397 DddAtox-N–SaKKH-Cas9(D10A)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7377 bp
- Type:
- Gene expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Promoter:
- CMV
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
G1397 DddAtox-N–SaKKH-Cas9(D10A) vector Vector Map
Plasmid Resuspension 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 ℃.
G1397 DddAtox-N–SaKKH-Cas9(D10A) vector Sequence
LOCUS 62056_951 7377 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 7377) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..7377 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 835..3990 /codon_start=1 /label=SaCas9 /note="Cas9 endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system" /translation="KRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF ITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDN DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKG" CDS 4024..4089 /codon_start=1 /label=3xFLAG /note="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4102..4350 /codon_start=1 /label=UGI /note="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 4363..4383 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 4392..4409 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" polyA_signal 4438..4662 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4733..4749) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4757..4773) /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)." promoter complement(4781..4811) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(4826..4847) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5135..5723) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5897..6754) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6755..6859) /label=AmpR promoter enhancer 7129..7377 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer"
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