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。
Introducing an inducible expression system into the groESL operon allows for co-expression of a pre-aggregation chaperonin (groESL) and a target protein (bgaB). This system utilizes IPTG to induce co-expression of 80% chaperonin and 20% target protein. Combining this system with the chaperonin-encoding genes htpG and pbpE resulted in 10-13% induced chaperonin. The method was further extended to co-express and identify the amyQα-amylase and the A and B subunits of glutamate synthase using Western blotting. To enhance expression, the chaperonin-protein complex was inserted into a high-copy SD plasmid with multiple cloning sites. The amyQ gene region of α-amylase was inserted into the SD plasmid cloning region, yielding pHT01, which facilitated the expression of a soluble chaperonin-protein complex.
- 载体名称:
- pHT43
- 载体抗性:
- Ampicillin, Chloramphenicol
- 载体长度:
- 8055 bp
- 载体类型:
- Bacillus subtilis expression vectors
- 复制子:
- ori
- 拷贝数:
- High copy number
- 启动子:
- Pgrac
- 克隆方法:
- Enzyme digestion and ligation
- 5'引物:
- LacI-R：GGCATACTCTGCGACATCGT
- 载体标签:
- N-amyQ
- 感受态:
- stbl3
- 培养温度:
- 37℃
pHT43 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。
pHT43 载体序列
LOCUS Exported 8055 bp DNA circular SYN 13-DEC-2024
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pHT43
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8055)
AUTHORS 11111111
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8055
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 2932..2957
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 3021..3284
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 3982..5016
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 5392..5898
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(111..761)
/codon_start=1
/label=Cm
/translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
primer_bind 1236..1252
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(1295..2377)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKTAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
protein_bind 2761..2785
/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)."
sig_peptide 2807..2899
/codon_start=1
/product="ǿźģTAбΪ棺
GCA TCA GCT"
/label=amyQ signal peptide (SPamyQ)
/translation="MIQKRKRTVSFRLVLMCTLLFVSLPITKTSA"
terminator 2932..2957
/label=trp terminator
CDS 3021..3284
/codon_start=1
/label=ParA family protein
/translation="MVETYEANIGLLGIVPVLIKSGGSVDQYILDEARKEFGDDLLNST
IKLRERIKRFDVQGITEEDTHDKEALKLFNNLTMELIERVEG"
CDS 3982..5016
/codon_start=1
/label=helix-turn-helix domain-containing protein
/translation="MSEVNLKGNTDELVYYRQQTTGNKIARKRIKKGKEEVYYVAETEE
KIWTEEQIKNFSLDKFGTHIPYIEGHYTILNNYFFDFWGYFLGAEGIALYAHLTRYAYG
SKDFCFPSLQTIAKKMDKTPVTVRGYLKLLERYGFIWKVNVRNKTKDNTEESPIFKIRR
KVPLLSEELLNGNPNIEIPDDEEAHVKKALKKEKEGLPKVLKKEHDEFVKKMMDESETI
NIPEALQYDTMYEDILSKGEIRKEIKKQIPNPTTSFESISMTTEEEKVDSTLKSEMQNR
VSKPSFDTWFKNTKIKIENKNCLLLVPSEFAFEWIKKRYLETIKTVLEEAGYVFEKIEL
RKVQ"
CDS complement(5392..5898)
/codon_start=1
/label=ORF-2
/translation="MPYSPTLLEGAVSDILLHKKILSPKDLRPEFLSKRFGIEIMTGKT
SYAYIDSDVKVFVLDKKVEEKERNYQFHKLFAHTLLHEENHLEIPMEVYEKHSEEAERL
TWVEAIPYHMLRYIDFSDPEFIKQASDVFYIPEKVVQNRINFMIQQQFEIDEFDNVNEK
VKSFA"
promoter 6132..6236
/gene="bla"
/label=AmpR promoter
CDS 6237..7097
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7268..7856
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"