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pcDNA3.1+/C-His 质粒 (编号: V011320)

Note: pcDNA3.1+/C-His vector is a mammalian expression vector with CMV promoter for expressing C-terminally 6xHis-tagged proteins.

pcDNA3.1+/C-His5432 bp60012001800240030003600420048005400CMV enhancerCMV promoterT7 promoterMCS6xHisbGH poly(A) signalf1 oriSV40 promoterNeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoter
复制序列 NovoBuilder中查看图谱

基本信息

载体名称:
pcDNA3.1+/C-His
载体抗性:
Ampicillin
载体长度:
5432 bp
载体类型:
Mammalian Expression Vectors
复制子:
ori
筛选标记:
Neomycin/G418(Geneticin)
拷贝数:
High copy number
启动子:
CMV
5'引物:
CTAGAGAACCCACTGCTTAC
3'引物:
TAGAAGGCACAGTCGAGG
载体标签:
C-term 6 x His
感受态:
Top10
培养温度:
37℃

产品信息

质粒编号:V011320
质粒名称:pcDNA3.1+/C-His
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。

参考文献

  • Ramalingam PS, Premkumar T, Sundararajan V, Hussain MS, Arumugam S. Design and development of dual targeting CAR protein for the development of CAR T-cell therapy against KRAS mutated pancreatic ductal adenocarcinoma using computational approaches. Discov Oncol. 2024 Oct 25;15(1):592. doi: 10.1007/s12672-024-01455-6. PMID: 39453574; PMCID: PMC11511808.

pcDNA3.1+/C-His 质粒 (编号: V011320)序列

LOCUS       pcDNA3.1+_C-His.        5432 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian expression vector with a CMV promoter for expressing 
            C-terminally 6xHis-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1+ C-His
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5432)
  AUTHORS   GenScript
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5432)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    895..985
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             986..1003
                     /label=6xHis
                     /note="6xHis affinity tag"
     polyA_signal    1029..1253
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1299..1727
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1741..2070
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2137..2928
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    3105..3238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3275..3291)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3299..3315)
                     /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(3323..3353)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3368..3389)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3677..4265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4439..5296)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5297..5401)
                     /label=AmpR promoter