pHapII vector (V005615)

COA reports Sequencing Results MSDS Download GenBank File(.gb)

Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005615	pHapII	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

The HpaII promoter was a strong promoter and promoted the high expression of laz in Gram-negative bacteria and the cat in B. subtilis.

Vector Name:: pHapII

Antibiotic Resistance:: Ampicillin

Length:: 6841 bp

Type:: GFP expression vector

Replication origin:: ori

Source/Author:: Zhang N, Wu K, He X, Li S-Q., Zhang Z-H., Shen B, Yang Y-M., Zhang R-F., Huang Q-W., Shen Q-R.

Growth Strain(s):: Top10

Growth Temperature:: 37℃

pHapII vector Map

Copy Sequence View Map

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

Zyprian E, Matzura H. Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system. DNA. 1986 Jun;5(3):219-25. doi: 10.1089/dna.1986.5.219. PMID: 3013549.

pHapII vector Sequence

LOCUS       Exported                6841 bp DNA     circular SYN 28-OCT-2024
DEFINITION  Shuttle expression-secretion vector pP43NMK, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6841)
  AUTHORS   Zhang XZ, Cui ZL, Hong Q, Li SP.
  TITLE     High-level expression and secretion of methyl parathion hydrolase in
            Bacillus subtilis WB800
  JOURNAL   Appl. Environ. Microbiol. 71 (7), 4101-4103 (2005)
  PUBMED    16000826
REFERENCE   2  (bases 1 to 6841)
  AUTHORS   Zhang X-Z., Cui Z-L., Li S-P.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-OCT-2005) Department of Microbiology, College of Life 
            Sciences, Nanjing Agricultural University, MOA Key Lab of 
            Microbiological Engineering of Agricultural Environment, #6 Tongwei 
            Road, Weigang, Nanjing, Jiangsu 210095, P. R. China
REFERENCE   3  (bases 1 to 6841)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6841)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6841)
  TITLE     Direct Submission
REFERENCE   6  (bases 1 to 6841)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
            Environ. Microbiol."; date: "2005"; volume: "71"; issue: "7"; pages:
            "4101-4103"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (25-OCT-2005) Department of Microbiology, College of Life Sciences,
            Nanjing Agricultural University, MOA Key Lab of Microbiological
            Engineering of Agricultural Environment, #6 Tongwei Road, Weigang,
            Nanjing, Jiangsu 210095, P. R. China"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
COMMENT     SGRef: number: 5; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             814..1815
                     /label=repB
                     /note="RepB replication protein"
     CDS             1987..2748
                     /codon_start=1
                     /gene="km"
                     /product="Km"
                     /label=km
                     /protein_id="ABD24446.1"
                     /translation="MNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTD
                     GPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFF
                     SILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTTFLP
                     SLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEK
                     LLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     gene            1987..2748
                     /gene="km"
                     /label=km
     CDS             2965..3369
                     /codon_start=1
                     /gene="ble"
                     /product="Ble"
                     /label=ble
                     /protein_id="ABD24447.1"
                     /translation="MRMLQSIPALPVGDIKKSIGFYCDKLGFTLVHHEDGFAVLMCNEV
                     RIHLWEASDEGWRSRSNDSPVCTGAESFIAGTASCRIEVEGIDELYQHIKPLGILHPNT
                     SLKDQWWDERDFAVIDPDNNLISFFQQIKS"
     gene            2965..3217
                     /gene="ble"
                     /label=ble
     promoter        3589..3808
                     /label=Hpa II promoter
     RBS             3859..3867
     CDS             3876..4589
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4620..4636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4644..4660)
                     /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)."
     promoter        complement(4668..4698)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4713..4734)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5022..5610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5784..6641)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6642..6746)
                     /label=AmpR promoter