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pCRY2PHR-mCherryN1 vector (V001069)

Price Information

Cat No. Plasmid Name Availability Add to cart
V001069 pCRY2PHR-mCherryN1 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:
pCRY2PHR-mCherryN1
Antibiotic Resistance:
Kanamycin
Length:
6188 bp
Type:
Mammalian Expression
Replication origin:
ori
Copy Number:
High Copy
Promoter:
CMV
Cloning Method:
Restriction Enzyme

pCRY2PHR-mCherryN1 vector Map

Copy Sequence View Map
pCRY2PHR-mCherryN16188 bp30060090012001500180021002400270030003300360039004200450048005100540057006000CMV enhancerCMV promoterCRY2PHRmCherrySV40 poly(A) signalf1 oriAmpR promoterSV40 promoterNeoR/KanRTK-pA-RHSV TK poly(A) signalori

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.

pCRY2PHR-mCherryN1 vector Sequence

LOCUS       40924_13505        6188 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses CRY2(1-498)-mCherry fusion for use in light-inducible 
            protein interaction modules.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6188)
  AUTHORS   Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL
  TITLE     Rapid blue-light-mediated induction of protein interactions in 
            living cells.
  JOURNAL   Nat Methods. 2010 Oct 31. ():.
  PUBMED    21037589
REFERENCE   2  (bases 1 to 6188)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6188)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2010 Oct 31. ():."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             625..2118
                     /codon_start=1
                     /label=CRY2PHR
                     /note="N-terminal photolyase homology region of Arabidopsis
                     thaliana cryptochrome 2"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     CDS             2143..2850
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    2976..3097
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(3104..3559)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3586..3690
                     /label=AmpR promoter
     promoter        3692..4049
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4084..4875
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5066..5085)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5110..5157
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5486..6074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"