LOCUS       sequence-355851-        2303 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 2303)
  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 2303)
  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-3b
FEATURES             Location/Qualifiers
     source          1..2303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..272
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     24..44
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     195..214
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        294..369
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(496..1084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(576..595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1255..2046,2047..2115))
                     /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     1878..1897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2116..2220)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccggtaccaa ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata
       61 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      121 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      181 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      241 ctttatatat cttgtggaaa ggacgaaaca ccgattgcaa agattgctga ctagttttag
      301 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      361 agtcggtgcg caatgctgta gtcagcaatc ttttttttta agcttgggcc gctcgaggta
      421 cctctctaca tatgacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      661 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      901 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1081 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1141 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1201 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1261 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1321 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1381 tgcaatgata ccgcgagatc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1441 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1501 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1561 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1621 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1681 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1741 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1801 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1861 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1921 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1981 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2041 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2101 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2161 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2221 cacatttccc cgaaaagtgc cacctgacgt cgctagctgt acaaaaaagc aggctttaaa
     2281 ggaaccaatt cagtcgactg gat
//
