pSIL2 载体 (V003189)

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:
pSIL2
载体抗性:
Ampicillin
载体长度:
4507 bp
载体类型:
Bacterial expression vector
复制子:
ori
载体来源:
De Schamphelaire W, Olbrechts A, Meert J, Verhelst K, Roggeman Fonseca M, Vanhoucke M, Beyaert R.

pSIL2 载体图谱

pSIL24507 bp600120018002400300036004200fd terminatorfd terminatorf1 orioriISSCmRcat promotertac promoterlac operator5' UTR of ompA, incl. RBSOmpA signal peptideunnamed protein product; mIL2 mature sequence3' UTR of mIL2

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。

pSIL2 载体序列

LOCUS       40924_40347        4507 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Bacterial expression vector pSIL2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4507)
  AUTHORS   De Schamphelaire W, Olbrechts A, Meert J, Verhelst K, Roggeman 
            Fonseca M, Vanhoucke M, Beyaert R.
  TITLE     BCCM/LMBP Plasmid collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4507)
  AUTHORS   De Schamphelaire W.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-FEB-2017) BCCM/LMBP, Universiteit Gent, 
            Technologiepark 927, 9052, BELGIUM
REFERENCE   3  (bases 1 to 4507)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4507)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-FEB-2017) BCCM/LMBP, Universiteit Gent, Technologiepark 927, 
            9052, BELGIUM"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      27..75
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     regulatory      134..215
                     /label=fd terminator
                     /note="fd terminator"
                     /regulatory_class="terminator"
     terminator      137..185
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      313..768
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     rep_origin      complement(927..1515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(1984..2362)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene;
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     CDS             complement(2864..3520)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(3521..3623)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     promoter        3788..3816
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    3824..3840
                     /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)."
     misc_feature    3865..3881
                     /label=5' UTR of ompA, incl. RBS
                     /note="5' UTR of ompA, incl. RBS"
     sig_peptide     3882..3944
                     /label=OmpA signal peptide
                     /note="signal peptide from the E. coli outer membrane
                     protein OmpA"
     CDS             3948..4397
                     /codon_start=1
                     /note="unnamed protein product; mIL2 mature sequence"
                     /protein_id="SJL88730.1"
                     /translation="APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENY
                     RNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNI
                     RVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ"
     misc_feature    4398..4500
                     /label=3' UTR of mIL2
                     /note="3' UTR of mIL2"