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).
| 载体基本信息 | |||
| 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 |
pNZ8148 载体载体图谱
质粒转化方法:
1. Take MC1061 competent cells out of -80°C and thaw on ice.
2. Remove agar plates (containing 10 μg/ml chloramphenicol) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5. Heat shock the transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 90 secs.
6. Put the tubes back on ice for 5 min.
7. Add 250-1,000 μl LB (without antibiotic) to the bacteria and grow in 30°C shaking incubator for 1-1.5 hour.
8. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic. Growth of colonies may take 2 days.
pNZ8148 载体载体序列
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"
