LOCUS       sequence-350193-        3087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coselection for PE3 in human cells. Vector for tandem expression of 
            ATP1A1 G8 sgRNA with a user-specified PE3 nick sgRNA from two 
            independent U6 promoters. Cloning of oligos using BbsI sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3087)
  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 3087)
  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_G8_Dual_sgRNA
FEATURES             Location/Qualifiers
     source          1..3087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..269
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        291..366
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        485..733
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        752..827
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(936..952)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(936..952)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(949..971)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    960..976
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(984..990,991..1008,1009..1014))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1029..1050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1167..1184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1338..1926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1418..1437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2097..2888,2889..2957))
                     /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     2720..2739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2958..3062)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacagt gagggcctat ttcccatgat tccttcatat ttgcatatac
       61 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
      121 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      181 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      241 tatatatctt gtggaaagga cgaaacaccg cagtgcccaa gtcaatgcag gttttagagc
      301 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      361 cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta
      421 gcgcgtgcgc caattctgca gacaaatggc gtcgactgga tccggtacca aggtcgggca
      481 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
      541 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      601 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      661 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      721 aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag caagttaaaa
      781 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
      841 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
      901 agacaaatgg ctctagtaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
      961 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1021 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1081 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1141 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1201 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1261 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1321 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1381 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1441 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1501 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1561 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1621 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1681 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1741 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1801 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1861 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1921 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     1981 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2041 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2101 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2161 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2221 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2281 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2341 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2401 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2461 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2521 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2581 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2641 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2701 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2761 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2821 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2881 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     2941 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3001 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3061 cgcacatttc cccgaaaagt gccacct
//
