Note: A-to-G base editor
基本信息
- 载体名称:
- ABE8e
- 载体抗性:
- Ampicillin
- 载体长度:
- 8217 bp
- 载体类型:
- CRISPR Plasmids
- 载体来源:
- David Liu
- 启动子:
- CMV
- 感受态:
- Top10
- 培养温度:
- 37℃
产品信息
质粒编号:V017504
质粒名称:ABE8e
规格:5 μg (冻干粉)
下载资源
我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
确保质粒的关键元件正确，但是我们并不能保证实验效果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
ABE8e 质粒 (编号: V017504)序列
LOCUS Exported 8217 bp DNA circular SYN 25-SEP-2024
DEFINITION A-to-G base editor.
ACCESSION .
VERSION .
KEYWORDS ABE8e
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8217)
AUTHORS Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW,
Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR
TITLE Phage-assisted evolution of an adenine base editor with improved Cas
domain compatibility and activity.
JOURNAL Nat Biotechnol. 2020 Jul;38(7):883-891. doi:
10.1038/s41587-020-0453-z. Epub 2020 Mar 16.
PUBMED 32433547
REFERENCE 2 (bases 1 to 8217)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/s41587-020-0453-z"; journalName: "Nat Biotechnol"; date:
"2020-07"; volume: "38"; issue: "7"; pages: "883-891"
FEATURES Location/Qualifiers
source 1..8217
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 168..371
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 322..342
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 413..432
/label=T7
/note="T7 promoter, forward primer"
promoter 413..431
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 481..501
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1096..5196
/codon_start=1
/product="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/label=Cas9(D10A)
/note="generates RNA-guided single strand nicks in DNA due
to the D10A mutation in the RuvC catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5239..5259
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5268..5285
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5308..5325)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5314..5538
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(5609..5625)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5609..5625)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5622..5644)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5633..5649
/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(5657..5687)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5702..5723
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5840..5857)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6011..6599)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6091..6110)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6770..7630)
/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"
primer_bind 7393..7412
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7631..7735)
/gene="bla"
/label=AmpR promoter
primer_bind complement(7814..7833)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer join(8005..8217,1..167)
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"