LOCUS       sequence-350131-        3089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control vector for coselection for PE3 in human cells. Tandem 
            expression of ATP1A1 G8 and MTOR Nick exon-44 sgRNAs from two 
            independent U6 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3089)
  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 3089)
  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_MTOR_Nick_Exon-44_Dual_sgRNA
FEATURES             Location/Qualifiers
     source          1..3089
                     /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        754..829
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(938..954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(938..954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(951..973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    962..978
                     /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(986..992,993..1010,1011..1016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1031..1052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1169..1186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1340..1928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1420..1439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2099..2890,2891..2959))
                     /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     2722..2741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2960..3064)
                     /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 accgtggcat gaaggcctgg aaggttttag agctagaaat agcaagttaa
      781 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
      841 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct
      901 gcagacaaat ggctctagta agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
      961 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1021 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1741 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3061 cgcgcacatt tccccgaaaa gtgccacct
//
