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pHT01 vector (V001452)

Price Information

Cat No. Plasmid Name Availability Add to cart
V001452 pHT01 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 pHT01 is a E. coli - B. subtilis shuttle vector.

Vector Name:
pHT01
Antibiotic Resistance:
Ampicillin, Chloramphenicol
Length:
7956 bp
Type:
Bacillus subtilis expression vectors
Replication origin:
ori
Promoter:
grac
Growth Strain(s):
Top10
Growth Temperature:
37℃

pHT01 vector Map

Copy Sequence View Map
pHT017956 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900720075007800Chloramphenicol acetyltransferaseM13 fwdlacIPgractrp terminatorParA family proteinhelix-turn-helix domain-containing proteinORF-2AmpR 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.

References

  • Babar TK, Glare TR, Hampton JG, Hurst MRH, Narciso J, Sheen CR, Koch B. Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates. Appl Microbiol Biotechnol. 2023 Jul;107(13):4337-4353. doi: 10.1007/s00253-023-12563-8. Epub 2023 May 19. PMID: 37204448; PMCID: PMC10313851.

pHT01 vector Sequence

LOCUS       Exported                7956 bp DNA     circular SYN 27-AUG-2024
DEFINITION  Exported.
ACCESSION   V001452
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7956)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7956)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7956)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(116..763)
                     /gene="cat"
                     /label=Chloramphenicol acetyltransferase
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     primer_bind     1239..1255
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1301..2380)
                     /label=lacI
                     /note="lac repressor"
     promoter        2684..2803
                     /label=Pgrac
     protein_bind    2764..2788
                     /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)."
     terminator      2833..2858
                     /label=trp terminator
     CDS             2922..3185
                     /codon_start=1
                     /label=ParA family protein
                     /translation="MVETYEANIGLLGIVPVLIKSGGSVDQYILDEARKEFGDDLLNST
                     IKLRERIKRFDVQGITEEDTHDKEALKLFNNLTMELIERVEG"
     CDS             3883..4917
                     /codon_start=1
                     /label=helix-turn-helix domain-containing protein
                     /translation="MSEVNLKGNTDELVYYRQQTTGNKIARKRIKKGKEEVYYVAETEE
                     KIWTEEQIKNFSLDKFGTHIPYIEGHYTILNNYFFDFWGYFLGAEGIALYAHLTRYAYG
                     SKDFCFPSLQTIAKKMDKTPVTVRGYLKLLERYGFIWKVNVRNKTKDNTEESPIFKIRR
                     KVPLLSEELLNGNPNIEIPDDEEAHVKKALKKEKEGLPKVLKKEHDEFVKKMMDESETI
                     NIPEALQYDTMYEDILSKGEIRKEIKKQIPNPTTSFESISMTTEEEKVDSTLKSEMQNR
                     VSKPSFDTWFKNTKIKIENKNCLLLVPSEFAFEWIKKRYLETIKTVLEEAGYVFEKIEL
                     RKVQ"
     CDS             complement(5293..5799)
                     /codon_start=1
                     /label=ORF-2
                     /translation="MPYSPTLLEGAVSDILLHKKILSPKDLRPEFLSKRFGIEIMTGKT
                     SYAYIDSDVKVFVLDKKVEEKERNYQFHKLFAHTLLHEENHLEIPMEVYEKHSEEAERL
                     TWVEAIPYHMLRYIDFSDPEFIKQASDVFYIPEKVVQNRINFMIQQQFEIDEFDNVNEK
                     VKSFA"
     promoter        6033..6137
                     /label=AmpR promoter
     CDS             6138..6995
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7169..7757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"