LOCUS       sequence-367465-        3438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for prime editing in human cells. Vector for tandem 
            expression of ATP1A1 T804N-G6 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_G6_T804N_Std_Dual_epegRNA_tevopreQ1
FEATURES             Location/Qualifiers
     source          1..3438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        68..316
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     terminator      complement(326..357)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(374..1051)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1058..1069
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1087..1105
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1112..1130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1278..1866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1358..1377)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2037..2828,2829..2897))
                     /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     2660..2679
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2898..3002)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3065..3313
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3334..3409
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tttaagcttg ggccgctcga ggtacctctc tacgtcgact ggatccggta ccaaggtcgg
       61 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
      121 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
      181 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
      241 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
      301 gaaaggacga aacacccgag accgtagtca aaagcctccg gtcggaggct tttgacttgg
      361 ctgaggagtg ccattaagca ccggtggagt gacgaccttc agcacgttcg tactgttcaa
      421 cgatggtgta gtcttcgttg tgggaggtga tgtccagttt gatgtcggtt ttgtaagcac
      481 ccggcagctg aaccggtttt ttagccatgt aggtggtttt aacttcagcg tcgtagtgac
      541 caccgtcttt cagtttcaga cgcattttga tttcaccttt cagagcaccg tcttccgggt
      601 acatacgttc ggtggaagct tcccaaccca tggttttttt ctgcataacc ggaccgtcgg
      661 acgggaagtt ggtaccacgc agtttaactt tgtagatgaa ctcaccgtct tgcagggagg
      721 agtcctgggt aacggtaaca acaccaccgt cttcgaagtt cataacacgt tcccatttga
      781 aaccttccgg gaaggacagt ttcaggtagt ccgggatgtc agccgggtgt ttaacgtaag
      841 ctttggaacc gtactggaac tgcggggaca ggatgtccca agcgaacggc agcggaccac
      901 ctttggtaac tttcagttta gcggtctggg taccttcgta cggacgacct tcaccttcac
      961 cttcgatttc gaactcgtga ccgttaacgg aaccttccat acgaactttg aaacgcatga
     1021 actctttgat aacgtcttcg ctactcgcca tagatccttt ctcctctttc agatccgtgc
     1081 tcagtatctc tatcactgat agggatgtca atctctatca ctgataggga ggtctcgcgc
     1141 ggttctatct agttacgcgt taaaccaact agaatttttt taagcttggg ccgctcgagg
     1201 tacctctcta catatgacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1381 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1441 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1501 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1621 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1681 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1741 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1801 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1861 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     1921 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     1981 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2041 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2101 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2161 gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag
     2221 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2281 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2341 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2401 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2461 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2521 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2581 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2641 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2701 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2761 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2821 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     2881 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     2941 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3001 cgcacatttc cccgaaaagt gccacctgac gtcgctagct gtacatacca aggtcgggca
     3061 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     3121 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     3181 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     3241 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     3301 aggacgaaac accgcaaaca ttccactacc actgttttag agctagaaat agcaagttaa
     3361 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg acattcccca
     3421 gaggtagtgg aatgtttt
//
