LOCUS       sequence-355853-        2307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transiently expressing a pegRNA to introduce LRRK2-G2019S mutation 
            in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2307)
  AUTHORS   Li H, Busquets O, Verma Y, Syed KM, Kutnowski N, Pangilinan GR, 
            Gilbert LA, Bateup HS, Rio DC, Hockemeyer D, Soldner F
  TITLE     Highly efficient generation of isogenic pluripotent stem cell models
            using prime editing.
  JOURNAL   Elife. 2022 Sep 7;11:e79208. doi: 10.7554/eLife.79208.
  PUBMED    36069759
REFERENCE   2  (bases 1 to 2307)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-09-7"; volume: "11:e79208. doi"; pages: " 
            10.7554/eLife.79208"
COMMENT     Sequence Label: pU6-pegRNA-LRRK2-G2019S-3d
FEATURES             Location/Qualifiers
     source          1..2307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..280
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     32..52
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     203..222
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        302..377
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(508..1096)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(588..607)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1267..2058,2059..2127))
                     /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     1890..1909
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2128..2232)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gactggatcc ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata
       61 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      121 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      181 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      241 tttcttggct ttatatatct tgtggaaagg acgaaacacc gattgcaaag attgctgact
      301 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      361 tggcaccgag tcggtgcgca atgctgtagt cagcaatctt tgcatttttt taagcttggg
      421 ccgctcgagg tacctctcta catatgacat gtgagcaaaa ggccagcaaa aggccaggaa
      481 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      541 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
      601 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
      661 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
      721 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
      781 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
      841 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
      901 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
      961 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1021 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1081 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1141 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1201 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1261 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1321 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1381 tggccccagt gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc
     1441 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1501 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1561 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     1621 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1681 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     1741 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     1801 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     1861 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     1921 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     1981 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     2041 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     2101 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     2161 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2221 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgctagct gtacaaaaaa
     2281 gcaggcttta aaggaaccaa ttcagtc
//
