质粒编号
质粒名称
规格
价格
V017496
pGERM
5 μg (冻干粉)
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学术研究使用，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。
Note:pGERM is a suicide vector plasmid designed for gene disruption in Bacteroidesspecies and some fungi. It contains selectable markers for both E. coli(e.g., ampicillin resistance gene, bla) and the target organism (e.g., erythromycin resistance gene, ermG, for Bacteroides). It also includes an origin of transfer (oriTfrom RK2) for conjugation and a multiple cloning site. Since it cannot replicate in Bacteroides, selection for erythromycin resistance allows for the identification of clones where the plasmid has integrated into the chromosome via homologous recombination, enabling targeted gene disruption.
- 载体名称:
- pGERM
- 载体抗性:
- Ampicillin
- 载体长度:
- 4807 bp
- 载体类型:
- Bacteroides Plasmid
- 载体来源:
- Shoemaker,N.B., Wang,G.R. and Salyers,A.A.
- 拷贝数:
- High copy number
- 感受态:
- stbl3
- 培养温度:
- 37℃
pGERM 载体图谱
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。
pGERM 载体序列
LOCUS pGERM 4807 bp DNA circular SYN 26-DEC-2025
DEFINITION PGERM gene disruption vector, complete sequence.
ACCESSION EF155418
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4807)
AUTHORS Shoemaker NB, Wang GR, Salyers AA.
TITLE Multiple gene products and sequences required for excision of the
mobilizable integrated Bacteroides element NBU1
JOURNAL J. Bacteriol. 182 (4), 928-936 (2000)
PUBMED 10648516
REFERENCE 2 (bases 1 to 4807)
AUTHORS Chiang HC, Manchester JK, Gordon JI.
TITLE Regulation of nitrogen metabolism by a novel hybrid two-component
system protein in a prominent human gut symbiont, Bacteroides
thetaiotaomicron
JOURNAL Unpublished
REFERENCE 3 (bases 1 to 4807)
AUTHORS Chiang HC, Manchester JK, Gordon JI.
TITLE Direct Submission
JOURNAL Submitted (03-DEC-2006) Center for Genome Sciences, Washington
University School of Medicine, 4444 Forest Park Ave. Campus Box
8510, Saint Louis, MO 63108, USA
REFERENCE 4 (bases 1 to 4807)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 4807)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Bacteriol."; date: "2000"; volume: "182"; issue: "4"; pages:
"928-936"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(03-DEC-2006) Center for Genome Sciences, Washington University
School of Medicine, 4444 Forest Park Ave. Campus Box 8510, Saint
Louis, MO 63108, USA"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4807
/mol_type="other DNA"
/label=pGERM is pUC19 with the RK2 oriT and ermG
/note="pGERM is pUC19 with the RK2 oriT and ermG"
/db_xref="taxon:433620"
/organism="pGERM gene disruption vector"
CDS complement(44..367)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/label=lacZ-alpha
/translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
DAA"
primer_bind 277..293
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 294..350
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(363..379)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 387..403
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(411..441)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 456..477
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(709..1080)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 1113..1224
/label=origin of transfer from 56 kb circular plasmid RK2
/note="origin of transfer from 56 kb circular plasmid RK2"
oriT 1113..1222
/label=incP origin of transfer
/note="incP origin of transfer"
rep_origin complement(1543..2131)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
gene complement(2302..3162)
/gene="AmpR"
/label=AmpR
CDS complement(2302..3162)
/codon_start=1
/transl_table=11
/gene="AmpR"
/product="beta lactamase"
/label=AmpR
/protein_id="ABO69572.1"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHKMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
gene 3650..4384
/gene="ermG"
/label=ermG
CDS 3650..4384
/codon_start=1
/transl_table=11
/gene="ermG"
/product="rRNA methyltransferase"
/label=ermG
/protein_id="ABO69573.1"
/translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
DINNISFEQFVSLFNSYKIFNG"