pGRG36pir vector (V005882)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005882	pGRG36pir	In stock, 1 week for quality controls	Buy one, get one free!

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

Basic Vector Information

pGRG36pir is similar to pGRG36, except that it contains a pir/Pi protein replicates vectors with an R6K origin.

Cells with those thermosensitive plasmids (pGRG36/pGRG36pir) should grown at 30 °C. Bacterial cultures were grown for no more than 18 h.

Vector Name:: pGRG36pir

Antibiotic Resistance:: Ampicillin

Length:: 13905 bp

Type:: Cloning vector, Tn7 delivery vector, Bacterial Exp

Replication origin:: pSC101 ori

Source/Author:: Kvitko BH, Bruckbauer S, Prucha J, McMillan I, Breland EJ, Lehman S, Mladinich K, Choi KH, Karkhoff-Schweizer R, Schweizer HP.

Copy Number:: Low copy number

Promoter:: araBAD

Growth Strain(s):: DH5alpha

Growth Temperature:: 30℃

pGRG36pir 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.

Note: DH5alpha in LB + ampicillin, grow at less than 32 degrees C

References

Kvitko BH, Bruckbauer S, Prucha J, McMillan I, Breland EJ, Lehman S, Mladinich K, Choi KH, Karkhoff-Schweizer R, Schweizer HP. A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids. BMC Res Notes. 2012 Mar 20;5:157.

Stoudenmire JL, Essock-Burns T, Weathers EN, Solaimanpour S, Mrázek J, Stabb EV. An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter. Appl Environ Microbiol. 2018 Jul 2;84(14):e00603-18.

pGRG36pir vector Sequence

LOCUS       V005882                13905 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V005882
VERSION     V005882
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 13905)
  AUTHORS   Kvitko BH, Bruckbauer S, Prucha J, McMillan I, Breland EJ, Lehman S,
            Mladinich K, Choi KH, Karkhoff-Schweizer R, Schweizer HP.
  TITLE     A simple method for construction of pir+ Enterobacterial hosts for
            maintenance of R6K replicon plasmids
  JOURNAL   BMC Res Notes 5, 157 (2012)
   PUBMED   22433797
REFERENCE   2  (bases 1 to 13905)
  AUTHORS   Bruckbauer ST.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JUN-2012) Microbiology Immunology and Pathology,
            Colorado State University, IDRC at Foothills Campus, Campus Delivery
            0922, Fort Collins, CO 80523-0922, USA
REFERENCE   3  (bases 1 to 13905)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 13905)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Res
            Notes 5, 157 (2012)"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (25-JUN-2012) Microbiology Immunology and Pathology, Colorado State
            University, IDRC at Foothills Campus, Campus Delivery 0922, Fort
            Collins, CO 80523-0922, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..13905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(149..1024)
                     /label="araC"
                     /note="L-arabinose regulatory protein"
     promoter        1051..1335
                     /label="araBAD promoter"
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite
                     direction (Guzman et al., 1995)"
     CDS             1453..2271
                     /gene="tnsA"
                     /label="Transposon Tn7 transposition protein TnsA"
                     /note="Transposon Tn7 transposition protein TnsA from
                     Escherichia coli. Accession#: P13988"
     CDS             2261..4366
                     /gene="tnsB"
                     /label="Transposon Tn7 transposition protein TnsB"
                     /note="Transposon Tn7 transposition protein TnsB from
                     Escherichia coli. Accession#: P13989"
     CDS             4366..6030
                     /gene="tnsC"
                     /label="Transposon Tn7 transposition protein TnsC"
                     /note="Transposon Tn7 transposition protein TnsC from
                     Escherichia coli. Accession#: P05846"
     CDS             6036..7562
                     /codon_start=1
                     /gene="tnsD"
                     /product="TnsD"
                     /label="tnsD"
                     /note="Tn7 transposase D"
                     /protein_id="AFR24693.1"
                     /translation="MRNFPVPYSNELIYSTIARAGVYQGIVSPKQLLDEVYGNRKVVAT
                     LGLPSHLGVIARHLHQTGRYAVQQLIYEHTLFPLYAPFVGKERRDEAIRLMEYQAQGAV
                     HLMLGVAASRVKSDNRFRYCPDCVALQLNRYGEAFWQRDWYLPALPYCPKHGALVFFDR
                     AVDDHRHQFWALGHTELLSDYPKDSLSQLTALAAYIAPLLDAPRAQELSPSLEQWTLFY
                     QRLAQDLGLTKSKHIRHDLVAERVRQTFSDEALEKLDLKLAENKDTCWLKSIFRKHRKA
                     FSYLQHSIVWQALLPKLTVIEALQQASALTEHSITTRPVSQSVQPNSEDLSVKHKDWQQ
                     LVHKYQGIKAARQSLEGGVLYAWLYRHDRDWLVHWNQQHQQERLAPAPRVDWNQRDRIA
                     VRQLLRIIKRLDSSLDHPRATSSWLLKQTPNGTSLAKNLQKLPLVALCLKRYSESVEDY
                     QIRRISQAFIKLKQEDVELRRWRLLRSATLSKERITEEAQRFLEMVYGEE"
     gene            6036..7562
                     /gene="tnsD"
                     /label="tnsD"
     terminator      8196..8282
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      8374..8401
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        8420..8511
                     /label="AmpR promoter"
     CDS             8512..9369
                     /label="AmpR"
                     /note="beta-lactamase"
     oriT            complement(9543..9652)
                     /direction=LEFT
                     /label="oriT"
                     /note="incP origin of transfer"
     rep_origin      10392..10614
                     /label="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             10662..11609
                     /label="Rep101(Ts)"
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     mobile_element  12045..12210
                     /label="Tn7L"
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     protein_bind    12313..12337
                     /label="attB1"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             12472..13386
                     /gene="pir"
                     /label="PI protein"
                     /note="PI protein from Escherichia coli. Accession#:
                     P03067"
     protein_bind    complement(13603..13627)
                     /label="attB2"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    13694..13905
                     /label="Tn7R"
                     /note="Tn7R"