Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007080 | pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) | 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:
- pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9627 bp
- Type:
- Mammalian Expression, CRISPR ; in vitro transcript
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- oBK6928-CCAAGTCTCCACCCCATTGACG
- 3' Primer:
- oBK219-GGGAGTGGCACCTTCCAGGGTC
pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) 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.
pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) vector Sequence
LOCUS 40924_11795 9627 bp DNA circular SYN 12-JUL-2021
DEFINITION CMV and T7 promoter expression plasmid for human codon optimized
ABEmax(7.10) A-to-G base editor with
SpRY(D10A/A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R133
3P/R1335Q/T1337R) and P2A-EGFP.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9627)
AUTHORS Walton RT, Christie KA, Whittaker MN, Kleinstiver BP
TITLE Unconstrained genome targeting with near-PAMless engineered
CRISPR-Cas9 variants.
JOURNAL Science. 2020 Apr 17;368(6488):290-296. doi:
10.1126/science.aba8853. Epub 2020 Mar 26.
PUBMED 32217751
REFERENCE 2 (bases 1 to 9627)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9627)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aba8853"; journalName: "Science"; date:
"2020-04-17- 17"; volume: "368"; issue: "6488"; pages: "290-296"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9627
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(749..853)
/label=AmpR promoter
enhancer 1123..1502
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1503..1706
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 1748..1766
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1816..1836
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 1834..3024
/codon_start=1
/label=ABE(7.10)
/note="adenine base editor comprising wild-type E. coli
TadA fused to the evolved TadA*(7.10) mutant (Gaudelli et
al., 2017)"
/translation="VSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGW
NRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVF
GARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQ
SSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARD
EREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT
FEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA
ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGS"
CDS 3025..7125
/codon_start=1
/label=Cas9 VQR
/note="Streptococcus pyogenes Cas9 endonuclease with the
D1135V/R1335Q/T1337R mutations"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 7168..7188
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 7255..7971
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 7984..8004
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 8013..8030
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
polyA_signal 8059..8283
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(8354..8370)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(8378..8394)
/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(8402..8432)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8447..8468)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8756..9344)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(join(9518..9627,1..748))
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"