pAIDA1 vector (V012813)

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V012813	pAIDA1	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

A construct with the AIDA surface expression system for display of a passenger protein flanked by His and Myc tags

Vector Name:: pAIDA1

Antibiotic Resistance:: Chloramphenicol

Length:: 5561 bp

Type:: E.coli expression plasmid

Replication origin:: p15A ori

Source/Author:: Gen Larsson

Copy Number:: Low copy number

Promoter:: lacUV5

Cloning Method:: Enzyme Cut

5' Primer:: GAGCGGATAACAATTTCACACAGG

Growth Strain(s):: DH5alpha

Growth Temperature:: 37℃

pAIDA1 vector Vector Map

View Map in NovoBuilder

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

Jarmander J, Gustavsson M, Do TH, Samuelson P, Larsson G. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. Microb Cell Fact. 2012 Sep 3;11:118.

pAIDA1 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       pAIDA1.        5561 bp DNA     circular SYN 11-MAY-2023
DEFINITION  A construct with the AIDA surface expression system for display of a
            passenger protein flanked by His and Myc tags.
ACCESSION   .
VERSION     .
KEYWORDS    pAIDA1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5561)
  AUTHORS   Jarmander J, Gustavsson M, Do TH, Samuelson P, Larsson G
  TITLE     A dual tag system for facilitated detection of surface expressed 
            proteins in Escherichia coli.
  JOURNAL   Microb Cell Fact. 2012 Sep 3;11:118. doi: 10.1186/1475-2859-11-118.
  PUBMED    22943700
REFERENCE   2  (bases 1 to 5561)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Microb Cell
            Fact."; date: "2012-09-3"; volume: "11:118. doi"; pages: " 
            10.1186/1475-2859-11-118"
FEATURES             Location/Qualifiers
     source          1..5561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..31
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     protein_bind    39..55
                     /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)."
     CDS             77..229
                     /codon_start=1
                     /label=SP
                     /note="SP"
                     /translation="MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAVVSTIG
                     NAFAVD"
     CDS             230..247
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             248..271
                     /codon_start=1
                     /label=HRV3C
                     /note="HRV3C"
                     /translation="LEALFQGP"
     CDS             299..319
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             320..349
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             356..1849
                     /codon_start=1
                     /label=ADIA
                     /note="ADIA"
                     /translation="VNNNGSIVINNSIINGNITNDADLSFGTAKLLSATVNGSLVNNKN
                     IILNPTKESAAAIGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLVVKGNTSGQSDIV
                     YVNEDGSGGQTRDGINIISVEGNSDAEFSLKNRVVAGAYDYTLQKGNESGTDNKGWYLT
                     SHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITG
                     GISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTSN
                     KAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLT
                     ASAGGGYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDTHQEDNGTVVQGAGKNNIQ
                     TKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIK
                     TGIEGVITQNLSVNGGVAYQAGGHGSNAISGALGIKYSF"
     rep_origin      2344..2889
                     /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."
     primer_bind     3006..3023
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3473..3492)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3659..4315
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        4331..4408
                     /label=lacI promoter
     CDS             4409..5488
                     /label=lacI
                     /note="lac repressor"