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:
- v3em-Cterm-PE6c-nls-P2A-Ubvs-dualU6-Rosa26-twinPE-attB
- 载体抗性:
- Ampicillin
- 载体长度:
- 8235 bp
- 载体类型:
- Genome editing
- 复制子:
- ori
- 宿主:
- Adeno-associated virus
- 启动子:
- CBh
- 5'引物:
- v3em-Cterm-PE6c-dualU6-Rosa26-twinPE-attB
- 3'引物:
- G33180-F1
- 培养温度:
- 37℃
v3em-Cterm-PE6c-nls-P2A-Ubvs-dualU6-Rosa26-twinPE-attB 载体载体图谱
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。
v3em-Cterm-PE6c-nls-P2A-Ubvs-dualU6-Rosa26-twinPE-attB 载体载体序列
LOCUS 62056_23460 8235 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8235) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..8235 /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin 1..589 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 877..898 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 913..943 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 951..967 /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)." primer_bind 975..991 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" enhancer 1170..1455 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 1457..1734 /label=chicken beta-actin promoter CDS 2016..2036 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 3231..3251 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 4731..4751 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" CDS 4761..4787 /codon_start=1 /label=c-myc NLS /note="nuclear localization signal of human c-Myc proto-oncogene (Dang and Lee, 1988)" /translation="PAAKRVKLD" CDS 4797..4853 /codon_start=1 /label=P2A /note="2A peptide from porcine teschovirus-1 polyprotein" /translation="ATNFSLLKQAGDVEENPGP" CDS 4896..4916 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" promoter complement(5524..5837) /label=U6 promoter /note="RNA polymerase III promoter for mouse U6 snRNA (Das et al., 1988)" misc_RNA complement(5944..6019) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(6049..6289) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(6461..6477) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 6619..7074 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 7100..7204 /label=AmpR promoter CDS 7205..8062 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW"