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。