LOCUS       sequence-339522-        2709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control vector for coselection for prime editing in human cells. 
            Tandem expression of ATP1A1 Q118R-G4 and RUNX1 +1 ATG insertion 
            pegRNAs from two independent U6 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2709)
  AUTHORS   Levesque S, Mayorga D, Fiset JP, Goupil C, Duringer A, Loiselle A, 
            Bouchard E, Agudelo D, Doyon Y
  TITLE     Marker-free co-selection for successive rounds of prime editing in 
            human cells.
  JOURNAL   Nat Commun. 2022 Oct 7;13(1):5909. doi: 10.1038/s41467-022-33669-z.
  PUBMED    36207338
REFERENCE   2  (bases 1 to 2709)
  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: "2022-10-7"; volume: "13(1):5909. doi"; pages: " 
            10.1038/s41467-022-33669-z"
COMMENT     Sequence Label: ATP1A1_G4_Q118R_RUNX1_+1_ATGins_Dual_pegRNA
FEATURES             Location/Qualifiers
     source          1..2709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(482..1070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(562..581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1241..2032,2033..2101))
                     /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     1864..1883
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2102..2206)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2269..2517
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2538..2613
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg cattttcagg aggaagcgag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctgtct gaagccatcc
      361 atgcttcctc ctgaaaattt tttttaagct tgggccgctc gaggtacctc tctacatatg
      421 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      481 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      541 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      601 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      661 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      721 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      781 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      841 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      901 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      961 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1021 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1081 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1141 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1201 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1261 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1321 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1381 gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1441 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1501 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1561 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1621 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1681 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1741 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1801 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1861 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1921 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1981 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2041 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2101 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2161 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2221 aagtgccacc tgacgtcgct agctgtacat accaaggtcg ggcaggaaga gggcctattt
     2281 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     2341 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     2401 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     2461 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtt
     2521 cctcttctgt agcagctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2581 tatcaacttg aaaaagtggc accgagtcgg tgctagcatc agagctgcta cagaagagtt
     2641 tttttaagct tgggccgctc gaggtacctc tctacgtcga ctggatccgg taccaaggtc
     2701 gggcaggaa
//
