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pACYC184 vector (V012555)

Price Information

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V012555 pACYC184 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 pACYC184 contqains the p15A origin of replication and a tetracycline resistance gene.

Vector Name:
pACYC184
Antibiotic Resistance:
Chloramphenicol
Length:
4245 bp
Type:
Cloning Vectors
Replication origin:
p15A ori
Source/Author:
New England Biolabs
Copy Number:
Medium copy number
Growth Strain(s):
DH10b
Growth Temperature:
37℃

pACYC184 vector Map

Copy Sequence View Map
pACYC1844245 bp600120018002400300036004200cat promoterp15A oritet promoterTcRCmR

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

  • Li G, Wang L, Zhang H, Luan Y, Sun Q, Duo L. Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae. Infect Drug Resist. 2023 Aug 24;16:5587-5598. doi: 10.2147/IDR.S421598. PMID: 37645559; PMCID: PMC10461740.

pACYC184 vector Sequence

LOCUS       40924_3976        4245 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid containing the p15A origin of replication and a tetracycline
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4245)
  AUTHORS   New England Biolabs
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4245)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Compatible with plasmids containing the colE1/pMB1/pBR322/pUC origin
            of replication.
FEATURES             Location/Qualifiers
     source          1..4245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(848..1393)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     promoter        1505..1533
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             1581..2768
                     /codon_start=1
                     /label=TcR
                     /note="tetracycline efflux protein"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     CDS             complement(join(3808..4245,1..219))
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"