AAVS1-Neo-M2rtTA 载体 (V006735)

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

AAVS1-Neo-M2rtTA10080 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000HA-LSAT2ANeoR/KanRPGK poly(A) signalCMV enhancerchicken beta-actin promoterchimeric intronrtTA-AdvancedSV40 poly(A) signalHA-RT7 promoterM13 fwdccdBAmpRoriCAP binding sitelac promoterlac operatorM13 revT3 promoter

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"