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。
- 载体名称:
- pAAVS1-Puro-DNR
- 载体抗性:
- Ampicillin
- 载体长度:
- 7507 bp
- 载体类型:
- CRISPR Plasmids
- 复制子:
- ori
- 载体来源:
- OriGene
- 拷贝数:
- High copy number
- 启动子:
- mPGK
pAAVS1-Puro-DNR 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。
pAAVS1-Puro-DNR 载体序列
LOCUS pAAVS1-Puro-DNR. 7507 bp DNA circular SYN 01-JAN-1980 DEFINITION Mammalian donor vector for transgene integration at the AAVS1 locus in the human genome. Use together with pCas-Guide-AAVS1. ACCESSION . VERSION . KEYWORDS pAAVS1-Puro-DNR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7507) AUTHORS OriGene TITLE Direct Submission REFERENCE 2 (bases 1 to 7507) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..7507 /mol_type="other DNA" /organism="synthetic DNA construct" protein_bind complement(119..135) /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(143..173) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(188..209) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(497..1085) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1259..2116) /label=AmpR /note="beta-lactamase" promoter complement(2117..2221) /label=AmpR promoter rep_origin complement(2503..2958) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 3171..3187 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 3278..3779 /label=HA-L /note="left homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene" protein_bind complement(3792..3824) /label=loxP /note="Cre-mediated recombination occurs in the 8-bp core sequence (ATGTATGC) (Shaw et al., 2021)." polyA_signal complement(3851..4304) /label=PGK poly(A) signal /note="mouse phosphoglycerate kinase 1 polyadenylation signal" CDS complement(4384..4980) /label=PuroR /note="puromycin N-acetyltransferase" promoter complement(5020..5519) /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" enhancer 5637..5940 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5941..6144 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 6170..6188 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 6197..6300 /label=MCS /note="MCS" /note="multiple cloning site" CDS 6303..6332 /label=Myc /note="Myc (human c-Myc proto-oncogene) epitope tag" CDS 6351..6374 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" primer_bind complement(6394..6410) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" polyA_signal 6421..6897 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" misc_feature 6972..7501 /label=HA-R /note="right homology arm from the adeno-associated virus integration site (AAVS1) within intron 1 of the human PPP1R12C gene"