LOCUS       sequence-404708-        3041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Prime editing in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3041)
  AUTHORS   Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, 
            An M, Newby GA, Chen JC, Hsu A, Liu DR
  TITLE     Engineered pegRNAs improve prime editing efficiency.
  JOURNAL   Nat Biotechnol. 2021 Oct 4. pii: 10.1038/s41587-021-01039-7. doi: 
            10.1038/s41587-021-01039-7.
  PUBMED    34608327
REFERENCE   2  (bases 1 to 3041)
  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 
            Biotechnol. 2021 Oct 4. pii: 10.1038/s41587-021-01039-7. doi: 
            10.1038/s41587-021-01039-7."
COMMENT     Sequence Label: pU6-tevopreq1-GG-acceptor
FEATURES             Location/Qualifiers
     source          1..3041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(48..636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(128..147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(807..1598,1599..1667))
                     /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     1430..1449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1668..1772)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1879..2127
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1879..1899
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2050..2069
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     terminator      complement(2137..2168)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(2185..2862)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             2869..2880
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    2898..2916
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2923..2941
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
       61 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      721 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      781 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      841 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
      901 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga tccacgctca
      961 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1021 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1081 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1141 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1201 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1261 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1321 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1381 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1441 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1501 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1561 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1621 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1741 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     1801 gtcgctagct gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
     1861 accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
     1921 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     1981 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     2041 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     2101 tatatcttgt ggaaaggacg aaacacccga gaccgtagtc aaaagcctcc ggtcggaggc
     2161 ttttgacttg gctgaggagt gccattaagc accggtggag tgacgacctt cagcacgttc
     2221 gtactgttca acgatggtgt agtcttcgtt gtgggaggtg atgtccagtt tgatgtcggt
     2281 tttgtaagca cccggcagct gaaccggttt tttagccatg taggtggttt taacttcagc
     2341 gtcgtagtga ccaccgtctt tcagtttcag acgcattttg atttcacctt tcagagcacc
     2401 gtcttccggg tacatacgtt cggtggaagc ttcccaaccc atggtttttt tctgcataac
     2461 cggaccgtcg gacgggaagt tggtaccacg cagtttaact ttgtagatga actcaccgtc
     2521 ttgcagggag gagtcctggg taacggtaac aacaccaccg tcttcgaagt tcataacacg
     2581 ttcccatttg aaaccttccg ggaaggacag tttcaggtag tccgggatgt cagccgggtg
     2641 tttaacgtaa gctttggaac cgtactggaa ctgcggggac aggatgtccc aagcgaacgg
     2701 cagcggacca cctttggtaa ctttcagttt agcggtctgg gtaccttcgt acggacgacc
     2761 ttcaccttca ccttcgattt cgaactcgtg accgttaacg gaaccttcca tacgaacttt
     2821 gaaacgcatg aactctttga taacgtcttc gctactcgcc atagatcctt tctcctcttt
     2881 cagatccgtg ctcagtatct ctatcactga tagggatgtc aatctctatc actgataggg
     2941 aggtctcgcg cggttctatc tagttacgcg ttaaaccaac tagaattttt ttaagcttgg
     3001 gccgctcgag gtacctctct acatatgaca tgtgagcaaa a
//
