LOCUS       sequence-355850-        2301 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 2301)
  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 2301)
  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-3a
FEATURES             Location/Qualifiers
     source          1..2301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..288
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     40..60
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     211..230
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        310..385
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(510..1098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(590..609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1269..2060,2061..2129))
                     /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     1892..1911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2130..2234)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt tcccatgatt
       61 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      121 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      181 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      241 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga ttgcaaagat
      301 tgctgactag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      361 tgaaaaagtg gcaccgagtc ggtgcgcaat gctgtagtca gcaatctttt tttaagcttg
      421 ggccgctcga ggtacctctc tacatatgac atgtgagcaa aaggccagca aaaggccagg
      481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
      541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
      601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
      661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
      721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
      781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
      841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
      901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
      961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     1261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     1321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     1381 tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc agatttatca
     1441 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     1501 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     1561 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     1621 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     1681 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     1741 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     1801 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     1861 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     1921 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     1981 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2041 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2101 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2161 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2221 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgctag ctgtacaaaa
     2281 aagcaggctt taaaggaacc a
//
