pFB-LIC-Bse 载体 (V010743)

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:
pFB-LIC-Bse
载体抗性:
Ampicillin
载体长度:
6816 bp
载体类型:
Structural Genomics Vectors
复制子:
ori
载体来源:
Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown
拷贝数:
High copy number
启动子:
sacB

pFB-LIC-Bse 载体载体图谱

pFB-LIC-Bse6816 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600polyhedrin promoterATG6xHisTEV siteloxPsacB promoterSacBSV40 poly(A) signalTn7Lf1 oriAmpR promoterAmpRoriTn7RGmRPc 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。

pFB-LIC-Bse 载体载体序列

LOCUS       pFB-LIC-Bse.        6816 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Baculovirus vector encoding an N-terminal 6xHis-TEV cassette and 
            ampicillin resistance plus a SacB negative selection marker, for 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pFB-LIC-Bse
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6816)
  AUTHORS   Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown 
            NA, Gileadi O.
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience.
  JOURNAL   J. Struct. Biol. 2010;172:3-13.
  PUBMED    20541610
REFERENCE   2  (bases 1 to 6816)
  AUTHORS   Structural Genomics Consortium
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6816)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Biol."; date: "2010"; volume: "172"; pages: "3-13"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with BseRI to 
            remove the SacB cassette, and treat with T4 DNA polymerase plus 
            dGTP.
FEATURES             Location/Qualifiers
     source          1..6816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     CDS             148..150
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             154..171
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             196..216
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     protein_bind    complement(246..279)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        294..739
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             740..2158
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     polyA_signal    2403..2537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  complement(2566..2731)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2915..3370
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3397..3501
                     /label=AmpR promoter
     CDS             3502..4359
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4533..5121
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     mobile_element  complement(5424..5648)
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5718..6248)
                     /label=GmR
                     /note="gentamycin acetyltransferase"
     promoter        complement(6437..6465)
                     /label=Pc promoter
                     /note="class 1 integron promoter"