v3em-Cterm-PE2max-∆RNaseH-nls-P2A-nls-Ubvs-dualU6 vector (Cat. No.: V015065)

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Basic Information

Note: v3em-Cterm-PE2max-∆RNaseH-nls-P2A-nls-Ubvs-dualU6 is a dual-AAV prime editing component of the v3em PE-AAV system (Davis et al. 2023): encodes the C-terminal half of PE2max (RNaseH domain deleted, with NLS) that reconstitutes the full editor via split-intein trans-splicing with the N-terminal component, plus additional regulatory elements and dual U6 sgRNA cassettes. Enables efficient in vivo prime editing in mouse brain, liver, and heart.

Name:: v3em-Cterm-PE2max-∆RNaseH-nls-P2A-nls-Ubvs-dualU6

Antibiotic Resistance:: Ampicillin

Length:: 8203 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Adeno-associated virus

Promoter:: CBh

5' Primer:: G33179-F1

Growth Temperature:: 37℃

$ 199.0

In stock, instant shipping

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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

Davis JR, et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nature Biotechnology. 2023;42(2):253–264 doi: 10.1038/s41587-023-01758-z

Marks RM, Scott O, Ivakine EA, Cohn RD. An optimized toolkit for prime editing. Nature Biotechnology. 2024;42(2):187–189 doi: 10.1038/s41587-023-02091-1

Chen PJ, Liu DR. Prime editing for precise and highly versatile genome manipulation. Nature Reviews Genetics. 2022;24(3):161–177 doi: 10.1038/s41576-022-00541-1

v3em-Cterm-PE2max-∆RNaseH-nls-P2A-nls-Ubvs-dualU6 vector (Cat. No.: V015065) Sequence

LOCUS       v3em-Cterm-PE2ma        8203 bp    DNA     circular SYN 07-SEP-2026
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8203)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8203)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      283..738
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        764..868
                     /label=AmpR promoter
     CDS             869..1726
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1900..2488
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2812..2842
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2850..2866
                     /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)."
     primer_bind     2874..2890
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        3069..3354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3356..3632
                     /label=chicken beta-actin promoter
     CDS             3914..3934
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             3935..4048
                     /codon_start=1
                     /label=Npu‑C intein
                     /translation="IKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCFN"
     CDS             4049..5071
                     /codon_start=1
                     /label=PE2max
                     /translation="EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
                     VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
                     GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV
                     KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP
                     EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN
                     IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG
                     D"
     CDS             5129..5149
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             5174..6661
                     /codon_start=1
                     /product="Moloney murine leukemia virus"
                     /label=RT ∆RNaseH
                     /translation="TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQ
                     APLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKP
                     GTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
                     SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQY
                     VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT
                     EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQ
                     QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL
                     DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMT
                     HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCL"
     CDS             6710..6730
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             6740..6766
                     /codon_start=1
                     /label=c-myc NLS
                     /note="nuclear localization signal of human c-Myc
                     proto-oncogene (Dang and Lee, 1988)"
                     /translation="PAAKRVKLD"
     CDS             6776..6832
                     /codon_start=1
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6875..6895
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             6896..7159
                     /codon_start=1
                     /label=ubiquitin variant
                     /translation="AASLNGAPLIKDPMLIFVKTLTGKTITLEVEPSDTIENVKAKIQD
                     KEGIPPDQQRLAFAGKSLEDGRTLSDYNILKDSKLHPLLRLR"
     promoter        complement(7412..7725)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das
                     et al., 1988)"
     misc_RNA        complement(7811..7886)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7916..8156)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"