| 载体基本信息 | |||
| 载体名称 | pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 9627 bp | 载体类型 | Mammalian Expression, CRISPR ; in vitro transcript |
| 拷贝数 | High Copy | 启动子 | CMV and T7 |
| 克隆方法 | Gibson Cloning | 5'引物 | oBK6928-CCAAGTCTCCACCCCATTGACG |
| 3'引物 | oBK219-GGGAGTGGCACCTTCCAGGGTC | ||
pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) 载体载体图谱
pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025) 载体载体序列
LOCUS Exported 9627 bp ds-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 pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP (RTW5025)
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)
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"
FEATURES Location/Qualifiers
source 1..9627
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 511..530
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(749..853)
/gene="bla"
/label=AmpR promoter
primer_bind complement(932..951)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
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"
primer_bind 1657..1677
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 1748..1767
/label=T7
/note="T7 promoter, forward primer"
promoter 1748..1766
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1816..1836
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3025..4740
/codon_start=1
/product="N-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(N)
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
CDS 7168..7188
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7198..7254
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7255..7971
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(7300..7321)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(7561..7580)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7908..7929
/label=EGFP-C
/note="EGFP, forward primer"
CDS 7984..8004
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8013..8030
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(8053..8070)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
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"
primer_bind complement(8354..8370)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8367..8389)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8378..8394
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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 8447..8468
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8585..8602)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8756..9344)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8836..8855)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(join(9515..9627,1..748))
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
我们不提供该载体，载体信息仅供查询和参考。