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pJYS3_ΔcrtYf vector (V008180)

Price Information

Cat No. Plasmid Name Availability Add to cart
V008180 pJYS3_ΔcrtYf In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pJYS3_ΔcrtYf
Antibiotic Resistance:
Kanamycin
Length:
11982 bp
Type:
Bacterial Expression, CRISPR ; Shuttle vector Cory
Replication origin:
pSC101 ori
3' Primer:
pBAD-R

pJYS3_ΔcrtYf vector Map

Copy Sequence View Map
pJYS3_ΔcrtYf11982 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000105001100011500pSC101 oriNeoR/KanRrrnB T2 terminatorrrnB T1 terminatorRep101

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.

pJYS3_ΔcrtYf vector Sequence

LOCUS       Exported               11982 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Constitutive transcription of FnCpf1 and crRNA of crtYf.
ACCESSION   .
VERSION     .
KEYWORDS    pJYS3_Delta-crtYf
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11982)
  AUTHORS   Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, 
            Li Y, Wang R, Yang S
  TITLE     CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum.
  JOURNAL   Nat Commun. 2017 May 4;8:15179. doi: 10.1038/ncomms15179.
  PUBMED    28469274
REFERENCE   2  (bases 1 to 11982)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-05-4"; volume: "8:15179. doi"; pages: " 
            10.1038/ncomms15179"
FEATURES             Location/Qualifiers
     source          1..11982
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             3671..4465
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3725..3744)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4335..4354
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      8504..8531
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      8623..8708
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(10965..11915)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      join(11963..11982,1..203)
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"