LOCUS       sequence-367517-        3438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for prime editing in human cells. Vector for tandem 
            expression of ATP1A1 Q118R-G4 pegRNA with a user-specified epegRNA. 
            pU6-tevopreq1-GG-acceptor-like plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3438)
  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 3438)
  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_Std_Dual_epegRNA_tevopreQ1
FEATURES             Location/Qualifiers
     source          1..3438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     terminator      complement(330..361)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(378..1055)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1062..1073
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1091..1109
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1116..1134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1282..1870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1362..1381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2041..2832,2833..2901))
                     /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     2664..2683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2902..3006)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3069..3317
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3338..3413
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tttttttaag cttgggccgc tcgaggtacc tctctacgtc gactggatcc ggtaccaagg
       61 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc cgagaccgta gtcaaaagcc tccggtcgga ggcttttgac
      361 ttggctgagg agtgccatta agcaccggtg gagtgacgac cttcagcacg ttcgtactgt
      421 tcaacgatgg tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc ggttttgtaa
      481 gcacccggca gctgaaccgg ttttttagcc atgtaggtgg ttttaacttc agcgtcgtag
      541 tgaccaccgt ctttcagttt cagacgcatt ttgatttcac ctttcagagc accgtcttcc
      601 gggtacatac gttcggtgga agcttcccaa cccatggttt ttttctgcat aaccggaccg
      661 tcggacggga agttggtacc acgcagttta actttgtaga tgaactcacc gtcttgcagg
      721 gaggagtcct gggtaacggt aacaacacca ccgtcttcga agttcataac acgttcccat
      781 ttgaaacctt ccgggaagga cagtttcagg tagtccggga tgtcagccgg gtgtttaacg
      841 taagctttgg aaccgtactg gaactgcggg gacaggatgt cccaagcgaa cggcagcgga
      901 ccacctttgg taactttcag tttagcggtc tgggtacctt cgtacggacg accttcacct
      961 tcaccttcga tttcgaactc gtgaccgtta acggaacctt ccatacgaac tttgaaacgc
     1021 atgaactctt tgataacgtc ttcgctactc gccatagatc ctttctcctc tttcagatcc
     1081 gtgctcagta tctctatcac tgatagggat gtcaatctct atcactgata gggaggtctc
     1141 gcgcggttct atctagttac gcgttaaacc aactagaatt tttttaagct tgggccgctc
     1201 gaggtacctc tctacatatg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1261 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1321 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1381 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1441 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1501 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1561 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1621 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1681 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1741 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1801 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1861 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1921 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1981 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2041 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2101 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2161 cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa
     2221 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2281 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2341 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2401 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2461 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2521 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2581 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2641 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2701 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2761 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2821 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2881 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2941 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3001 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgct agctgtacat accaaggtcg
     3061 ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     3121 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     3181 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     3241 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     3301 ggaaaggacg aaacaccgtt cctcttctgt agcagctgtt ttagagctag aaatagcaag
     3361 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctagcatc
     3421 agagctgcta cagaagag
//
