Edit-R HDR Plasmid Donor Components

Insert and backbone for HDR plasmid construction


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Plasmid backbone and fluorescence insert DNA fragments for construction of an Edit-R HDR donor plasmid

The Edit-R HDR Plasmid Donor Kit for insertion of EGFP or mKate2 using the HDR pathway is constructed from a universal plasmid backbone, the DNA insert, and two custom homology arms. Assembly of a custom insert plasmid donor utilizes the same strategy only substituting a user-provided DNA fragment for the insert sequence. These products include the EGFP or mKate2 insert and the plasmid backbone only.

Use and assembly of the donor plasmid requires design of two primer pairs for amplification of the 5′ and 3′ homology arms flanking the insertion site. These primers can be designed and ordered using the HDR Donor Designer.

Highlights

  • Fast and efficient assembly of the DNA donor plasmid
  • Validated protocols for design, assembly, and confirmation of the donor plasmid
  • Utilize the CRISPR Configurator for designing the crRNA to the cut site 
  • Seamless integration with the HDR Donor Designer for designing the primers to create the required 5′ and 3′ homology arms flanking the insertion site

 

Included Components & Amounts

Cat # Description Item # Amount
UK-008100-02-10 Edit-R HDR Plasmid Donor & Insert - EGFP    
  Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng
  Edit-R HDR insert - EGFP U-008100-01-250 250 ng
UK-008200-04-10 Edit-R HDR Plasmid Donor & Insert -mKate2    
  Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng
  Edit-R HDR Insert - mKate2 U-008200-01-250 250 ng
U-008300-01-1200 Edit-R HDR Plasmid Donor Backbone U-008300-01-1200 1200 ng

Note: The backbone and EGFP or mKate2 inserts are included with the HDR Plasmid Donor Kit; they may be ordered separately if more material is needed.

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
Edit-R™ mKate2 Insert Sequence 
 
LOCUS: Edit-R mKate2 Sequence         
714 bp DNA linear   
            
FEATURES                    Location/Qualifiers
N-terminal flexible linker    1..12
mKate2                            13..705
C-terminal flexible linker    706..714
 
ORIGIN      
1 gctggtggca gcgtgagcga gctgattaag gagaacatgc acatgaagct gtacatggag
61 ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
121 ggcacccaga ccatgagaat caaggcggtc gagggcggcc ctctcccctt cgccttcgac
181 atcctggcta ccagcttcat gtacggcagc aaaaccttca tcaaccacac ccagggcatc
241 cccgacttct ttaagcagtc cttccccgag ggcttcacat gggagagagt caccacatac
301 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
361 tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
421 acactcggct gggaggcctc caccgagacc ctgtaccccg ctgacggcgg cctggaaggc
481 agagccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttgaagacc
541 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac
601 agaagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg
661 gctgtggcca gatactgcga cctccctagc aaactggggc acagaggagg tagc
//
 
Edit-R™ EGFP Insert Sequence 
 
LOCUS Edit-R EGFP Sequence
735 bp DNA linear   
 
FEATURES                   Location/Qualifiers
N-terminal flexible linker   1..12
EGFP                              13..726
C-terminal flexible linker   727..735
 
ORIGIN      
1 gctggtggca gcgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
61 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
121 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
181 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
241 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
301 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
361 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
421 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
481 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
541 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
601 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
661 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
721 tacaagggag gtagc
//

 

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LMNA tagged at the N-terminus with EGFP in U2OS cells

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U2OS cells were transfected with DharmaFECT Duo, Cas9 mRNA, synthetic crRNA-tracrRNA targeting LMNA, and an EGFP donor plasmid specific to the N-terminus of LMNA containing ~1000 bps of homology per homology arm. Cells were passaged as needed, and imaged 7-days post transfection on an InCell 2200 high content fluorescent microscope.


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mKate2-SEC61B fusion in U2OS cells

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Creation of an mKate2 N-terminal fusion to the SEC61B gene, a component of protein transport in the endoplasmic reticulum. U2OS cells were transfected with 50 nM SEC61B-crRNA:tracrRNA, 25 nM Cas9 protein, 2 ng/µL SEC61B mKate2 HDR donor plasmid, and 0.3 µL/well DharmaFECT Duo and visualized on a fluorescent microscope seven days post-transfection. Note: Ribonucleoprotein (RNP) complex ratio is 2:1 for Cas9 protein transfection.


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Materials required for cloning HDR plasmid donors

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The Edit-R HDR plasmid donor is created through plasmid cloning and assembly of a fluorescent protein insert sequence, the cut vector backbone, and two homology arms generated with custom PCR primers to geneomc regions flanking the insertion site.


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Dharmacon™ Edit-R™ mKate2 knock-in donor

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Edit-R mKate2 plasmid donor map with important vector features. The example includes 500 bp homology arms for reference.


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Diagram of the plasmid donor cloning workflow

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Diagram of the plasmid donor cloning workflow including component ordering, homology arm amplification, and plasmid donor generation. Colors on the diagram indicate the origin of the DNA (dark blue = 5' homology arm, light blue = 3' homology arm, red = mKate2 fluorescent reporter sequence, orange = plasmid backbone). medium alone = media-only negative PCR control.


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Workflow to acquire mKate2 knock-in clonal cell line

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Workflow showing the major process steps and general timing necessary to generate a mKate2 knock-in clonal cell line.