pNZ8148 vector (V012804#)

Price Information

Two vials of lyophilized plasmid will be delivered, each vial is about 5µg.

Basic Vector Information

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).

pNZ8148 vector Vector Map

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

Plasmid Transformation Protocol:

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 vector Sequence

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"