AAVS1 pegRNA1 with trimmed attP vector (V017501) Gene synthesis in AAVS1 pegRNA1 with trimmed attP backbone

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V017501	AAVS1 pegRNA1 with trimmed attP	In stock, instant shipping

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

Basic Vector Information

pegRNA 1 plasmid used for PASSIGE-mediated Bxb1 attP installation

Vector Name:: AAVS1 pegRNA1 with trimmed attP

Antibiotic Resistance:: Ampicillin

Length:: 2368 bp

Type:: CRISPR Plasmids

Source/Author:: David Liu

Copy Number:: High copy number

Promoter:: U6

Growth Strain(s):: TOP10

Growth Temperature:: 37℃

AAVS1 pegRNA1 with trimmed attP 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.

References

Pandey S, Gao XD, Krasnow NA, et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat Biomed Eng. Published online June 10, 2024. doi:10.1038/s41551-024-01227-1

AAVS1 pegRNA1 with trimmed attP vector Sequence

LOCUS       Exported                2368 bp DNA     circular SYN 06-SEP-2024
DEFINITION  pegRNA 1 plasmid used for PASSIGE-mediated Bxb1 attP installation.
ACCESSION   .
VERSION     .
KEYWORDS    AAVS1 pegRNA1 with trimmed attP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2368)
  AUTHORS   Pandey S, Gao XD, Krasnow NA, McElroy A, Tao YA, Duby JE, Steinbeck 
            BJ, McCreary J, Pierce SE, Tolar J, Meissner TB, Chaikof EL, Osborn 
            MJ, Liu DR
  TITLE     Efficient site-specific integration of large genes in mammalian 
            cells via continuously evolved recombinases and prime editing.
  JOURNAL   Nat Biomed Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1.
  PUBMED    38858586
REFERENCE   2  (bases 1 to 2368)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Biomed 
            Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1."
FEATURES             Location/Qualifiers
     source          1..2368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     340..359
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(632..707)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(728..976)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(786..805)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(956..976)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1083..1187
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1188..2048
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1406..1425)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2219..2368,1..439)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"