质粒编号
质粒名称
规格
价格
V012804
pNZ8148
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。
pNZ8148 contains an origin of replication (ORI), the gene for the resistance to chloramphenicol, two genes for the replication proteins repA and repC, the nisin-inducible promoter (P nisA), and the transcription terminator (T). The gene, tagged with a Strep-tag II (STREP) followed by a stop codon (*), can be inserted between the necessary NcoI site and another endonuclease site from the multicloning site (MCS) such as PstI, SphI, KpnI, SpeI, XbaI, SacI and HindIII. The replicons of the vectors pNZ8008, pNZ8148, pNZ8149 and pNZ8150 are identical and came originally from the Lactococcus lactis plasmid pSH71. However, this replicon has a broad host range. Plasmids with this replicon can replicate in many Gram-positive bacteria, such as Lactobacillus plantarum and Streptococcus thermophilus. pNZ8148 – In this vector the nisA promoter is followed by an NcoI site for translational fusions at the ATG. It contains a terminator after the MCS. Sequence adaptation for cloning in NcoI can result in a change in the second amino acid of a protein (Mierau and Kleerebezem, 2005). For propagation of pNZ8148, we recommend E. coli strain MC1061 (10 µg/ml chloramphenicol; growth of colonies may take 2 days). pUC57-pNZ8148 is recommended as a replacement, which can be grown in conventional clonal strains and is easier to use.
- 载体名称:
- pNZ8148
- 载体抗性:
- Chloramphenicol
- 载体长度:
- 3167 bp
- 载体类型:
- expression plasmid of Lactobacilli
- 启动子:
- PnisA
- 克隆方法:
- Restriction Enzyme
- 感受态:
- MC1061
- 培养温度:
- 30℃
pNZ8148 载体图谱
质粒操作方法
Use MC1061 competent cells; 10 μg/ml chloramphenicol as antibiotics; Grow the colonies at 30°C and it may take 2 days.
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。
pNZ8148 载体序列
LOCUS Exported 3167 bp ds-DNA circular SYN 17-JUN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pNZ8148
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3167)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..3167
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 5..188
/note="Pnis"
/note="Nisin A promoter region"
terminator 393..445
/note="T"
CDS 788..997
/codon_start=1
/note="repC"
/note="Replication gene C"
/translation="MGGKEANFASVLRPPIKCRVPIFVPKTLYPNWLKGLRGFSIANES
PTFSPTFFINLYLSSFIFVFMITK"
CDS 1266..1964
/codon_start=1
/note="repA"
/note="Replication gene A"
/translation="MAIKNTKARNFGFLLYPDSIPNDWKEKLESLGVSMAVSPLHDMDE
KKDKDTWNSSDVIRNGKHYKKPHYHVIYIARNPVTIESVRNKIKRKLGNSSVAHVEILD
YIKGSYEYLTHESKDAIAKNKHIYDKKDILNINDFDIDRYITLDESQKRELKNLLLDIV
DDYNLVNTKDLMAFIRLRGAEFGILNTNDVKDIVSTNSSAFRLWFEGNYQCGYRASYAK
VLDAETGEIK"
CDS 2424..3074
/codon_start=1
/note="cm"
/note="chloramphenicol resistant gene"
/translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"