LOCUS       sequence-355852-        2305 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 2305)
  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 2305)
  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-3c
FEATURES             Location/Qualifiers
     source          1..2305
                     /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(506..1094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(586..605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1265..2056,2057..2125))
                     /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     1888..1907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2126..2230)
                     /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 tgttttttta agcttgggcc
      421 gctcgaggta cctctctaca tatgacatgt gagcaaaagg ccagcaaaag gccaggaacc
      481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      541 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      601 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      661 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      721 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      781 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      841 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      901 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      961 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1021 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1081 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1141 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1201 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1261 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1321 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1381 gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat ttatcagcaa
     1441 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1501 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1561 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1621 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1681 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1741 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1801 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1861 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1921 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1981 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2041 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2101 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2161 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2221 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgctagctgt acaaaaaagc
     2281 aggctttaaa ggaaccaatt cagtc
//
