LOCUS       sequence-357728-        2279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transiently expressing a nicking sgRNA to facilitate the 
            introduction of LRRK2-G2019S mutation in PE3 approach.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2279)
  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 2279)
  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: BPK1520-LRRK2-G2019S-ng
FEATURES             Location/Qualifiers
     source          1..2279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        112..360
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     112..132
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     283..302
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        381..456
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(560..1148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(640..659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1319..2110,2111..2179))
                     /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     1942..1961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(2180..2279,1..5))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgcgcacat ttccccgaaa agtgccacct gacgtcgcta gctgtacaaa aaagcaggct
       61 ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga agagggccta
      121 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga
      181 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      241 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      301 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      361 gacagacctg atcacctacc gttttagagc tagaaatagc aagttaaaat aaggctagtc
      421 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttaagcttg ggccgctcga
      481 ggtacctctc tacatatgac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      541 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      601 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      661 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      721 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      781 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      841 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      901 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      961 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1021 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1081 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1141 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1201 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     1261 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     1321 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1381 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1441 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1501 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1561 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1621 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1681 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1741 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1801 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1861 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1921 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1981 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2041 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2101 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2161 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     2221 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtt
//
