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。

The pK19mobsacB is a Corynebacterium glutamicum gene Knockout vector.

载体名称:
pK19mobsacB
载体抗性:
Kanamycin
载体长度:
5719 bp
载体类型:
Function E.coli Editing plasmids
复制子:
ori
载体来源:
Okamoto S, Niki H.
启动子:
sacB
感受态:
JM108
培养温度:
37℃

pK19mobsacB 载体载体图谱

pK19mobsacB5719 bp60012001800240030003600420048005400NeoR/KanRsacB promoterSacBoriTcat promotercat promoteroriCAP binding sitelac promoterlac operatorM13 revMCSM13 fwd

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。

pK19mobsacB 载体载体序列

LOCUS       40924_26496        5719 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pK19mobsacB DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5719)
  AUTHORS   Okamoto S, Niki H.
  TITLE     NBRP cloning vector collection sequence project
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5719)
  AUTHORS   Okamoto S, Niki H.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-APR-2017) Contact:Sho Okamoto National Institute of 
            Genetics, Microbial Genetics Laboratory; 1111 Yata, Mishima, 
            Shizuoka 411-8540, Japan URL 
            :https://www.nig.ac.jp/labs/MicroGen/index.html
REFERENCE   3  (bases 1 to 5719)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5719)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (07-APR-2017) Contact:Sho Okamoto National Institute of Genetics, 
            Microbial Genetics Laboratory; 1111 Yata, Mishima, Shizuoka 
            411-8540, Japan URL :https://www.nig.ac.jp/labs/MicroGen/index.html"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             346..1137
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        1186..1631
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             1632..3050
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            complement(3407..3516)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     promoter        complement(3751..3841)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     promoter        complement(4035..4125)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      4485..5073
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5361..5382
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5397..5427
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5435..5451
                     /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     5459..5475
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(5487..5543)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5544..5560)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"