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pNCMO2 vector (V017507) Gene synthesis in pNCMO2 backbone

Price Information

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V017507 pNCMO2 In stock, instant shipping

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

pNCMO2 is a shuttle vector for Brevibacillus and E. coli. Accordingly, the expression plasmid is constructed in E. coli, after which it is introduced to Brevibacillus for protein expression. The P2 promoter, derived from a cell wall protein of the host bacterium, is used as the expression promoter for pNCMO2. Because the P2 promoter does not work in E. coli, it is useful in the cloning of the target gene. However, it is an exceptionally strong promoter in Brevibacillus resulting in efficient protein production.

Vector Name:
pNCMO2
Antibiotic Resistance:
Ampicillin
Length:
5225 bp
Type:
Brevibacillus Expression Vector
Source/Author:
Takara Bio
Promoter:
P2
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pNCMO2 vector Map

Copy Sequence View Map
pNCMO25225 bp6001200180024003000360042004800P2 promoterLac operatorSecretion signal peptideRepBNmRAmpR promoterAmpRori

Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

pNCMO2 vector Sequence

LOCUS       Exported                5225 bp DNA     circular SYN 22-NOV-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pNCMO2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5225)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     Created by Takara Bio Inc. http://www.takarabio.com
            Cat. No. HB112
FEATURES             Location/Qualifiers
     source          1..5225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..103
                     /label=P2 promoter
     protein_bind    104..125
                     /label=Lac operator
                     /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)."
     misc_feature    258..347
                     /label=Secretion signal peptide
     misc_feature    346..409
                     /label=MCS
                     /note="multiple cloning site"
     CDS             926..1930
                     /codon_start=1
                     /label=RepB
                     /note="RepB replication protein from Enterococcus faecalis 
                     plasmid pAM-alpha-1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     CDS             2099..2860
                     /codon_start=1
                     /label=NmR
                     /note="Neomycin resistance gene, selection marker in 
                     Brevibacillus"
                     /translation="VNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTD
                     GPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFF
                     SILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTTFLP
                     SLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEK
                     LLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     promoter        3083..3187
                     /label=AmpR promoter
     CDS             3188..4048
                     /codon_start=1
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVPLIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4219..4807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"