LOCUS       sequence-368143-        2322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA plasmid in order to make a 5 basepair deletion at the HEK3 
            locus, uses an NGG-PAM site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2322)
  AUTHORS   Schene IF, Joore IP, Baijens JHL, Stevelink R, Kok G, Shehata S, 
            Ilcken EF, Nieuwenhuis ECM, Bolhuis DP, van Rees RCM, Spelier SA, 
            van der Doef HPJ, Beekman JM, Houwen RHJ, Nieuwenhuis EES, Fuchs SA
  TITLE     Mutation-specific reporter for optimization and enrichment of prime 
            editing.
  JOURNAL   Nat Commun. 2022 Mar 1;13(1):1028. doi: 10.1038/s41467-022-28656-3.
  PUBMED    35232966
REFERENCE   2  (bases 1 to 2322)
  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: "Nat 
            Commun."; date: "2022-03-1"; volume: "13(1):1028. doi"; pages: " 
            10.1038/s41467-022-28656-3"
COMMENT     Sequence Label: pegRNA-HEK3_del1-5-NGG
FEATURES             Location/Qualifiers
     source          1..2322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     430..449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(668..772)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        879..1127
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     879..899
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1050..1069
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1149..1224
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(1370..1958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1450..1469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2129..2322,1..598,599..667))
                     /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"
ORIGIN
        1 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
       61 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
      121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
      181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
      241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
      301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
      361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
      421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
      481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
      541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
      601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
      661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
      721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
      781 cccgaaaagt gccacctgac gtcgctagct gtacaaaaaa gcaggcttta aaggaaccaa
      841 ttcagtcgac tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc
      901 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
      961 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1021 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1081 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg cccagactga
     1141 gcacgtgagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     1201 gaaaaagtgg caccgagtcg gtgctggagg aagcagggct tcctttcctc tgccgtgctc
     1261 agtctgtttt tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa
     1321 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1381 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1441 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1501 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1561 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1621 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1681 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1741 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1801 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1861 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1921 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1981 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2041 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2101 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2161 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2221 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct
     2281 caccggctcc agatttatca gcaataaacc agccagccgg aa
//
