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pMA5 vector (V013300)

Price Information

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V013300 pMA5 In stock, instant shipping

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

Basic Vector Information

The pMA5 is an E. coli/B. subtilis shuttle plasmid which mainly used for protein expression in B. subtilis.

Vector Name:
pMA5
Antibiotic Resistance:
Ampicillin
Length:
7204 bp
Type:
Protein expression
Replication origin:
ori
Host:
Bacillus subtilis
Selection Marker:
Kan
Promoter:
HpaII
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pMA5 vector Map

Copy Sequence View Map
pMA57204 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200fd terminatorf1 oriAmpR promoterAmpRoriCAP binding sitecat promoterrepBkanamycin nucleotidyltransferasebleomycin binding protein

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.

References

  • Chen J, Fu G, Gai Y, Zheng P, Zhang D, Wen J. Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression. Microb Cell Fact. 2015;14:92. Published 2015 Jun 26. doi:10.1186/s12934-015-0282-9

  • Li T, Li H, Zhong L, Qin Y, Guo G, Liu Z, Hao N, Ouyang P. Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches. Microb Cell Fact. 2024 Feb 21;23(1):58. doi: 10.1186/s12934-024-02334-z. PMID: 38383407; PMCID: PMC10880289.

pMA5 vector Sequence

LOCUS       pMA5        7204 bp DNA     circular SYN 04-OCT-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7204)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      143..191
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      429..884
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1008..1112
                     /label=AmpR promoter
     CDS             1113..1970
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2144..2735
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    3023..3044
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        complement(3505..3607)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             4146..5147
                     /codon_start=1
                     /label=repB
                     /note="RepB replication protein"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     CDS             5310..6080
                     /codon_start=1
                     /label=kanamycin nucleotidyltransferase
                     /translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
                     QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHG
                     QFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTT
                     FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSD
                     SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     CDS             6303..6701
                     /codon_start=1
                     /label=bleomycin binding protein
                     /translation="MLQSIPALPVGDIKKSIGFYCDKLGFTLVHHEDGFAVLMCNEVRI
                     HLWEASDEGWRSRSNDSPVCTGAESFIAGTASCRIEVEGIDELYQHIKPLGILHPNTSL
                     KDQWWDERDFAVIDPDNNLISFFQQIKS"