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。
所有的产品都严格仅供科学研究使用,不能应用于临床试验,包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确,但是我们并不能保证实验结果。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。
AAVS1 donor vector for genomic targeting
- 载体名称:
- AAVS1-Neo-M2rtTA
- 载体抗性:
- Ampicillin
- 载体长度:
- 10080 bp
- 载体类型:
- Mammalian Expression Vectors
- 复制子:
- ori
- 筛选标记:
- Neomycin (select with G418)
- 启动子:
- CAG
AAVS1-Neo-M2rtTA 载体图谱
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。
AAVS1-Neo-M2rtTA 载体序列
LOCUS 40924_170 10080 bp DNA circular SYN 13-JAN-2022 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10080) TITLE Direct Submission REFERENCE 2 (bases 1 to 10080) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..10080 /mol_type="other DNA" /organism="synthetic DNA construct" misc_feature 33..836 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" misc_feature 843..868 /label=SA /note="splice acceptor site" CDS 892..945 /label=T2A /note="2A peptide from Thosea asigna virus capsid protein" CDS 961..1761 /label=NeoR/KanR /note="aminoglycoside phosphotransferase from Tn5" polyA_signal 1836..2291 /label=PGK poly(A) signal /note="mouse phosphoglycerate kinase 1 polyadenylation signal" enhancer 2389..2692 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 2694..2971 /note="chicken beta-actin promoter" intron 2973..3990 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" CDS 4090..4833 /label=rtTA-Advanced /note="improved tetracycline-controlled transactivator" polyA_signal complement(4846..4980) /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" misc_feature 5310..6146 /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" promoter complement(6187..6205) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6212..6228) /label=M13 fwd /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" CDS 6366..6665 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" CDS complement(8068..8925) /label=AmpR /note="beta-lactamase" rep_origin 9049..9637 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 9925..9946 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 9961..9991 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9999..10015 /label=lac repressor encoded by lacI binding site /bound_moiety="lac repressor encoded by lacI" /note="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)." primer_bind 10023..10039 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 10060..10078 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase"