LOCUS       sequence-340028-        3052 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 3052)
  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 3052)
  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-tmpknot-GG-acceptor
FEATURES             Location/Qualifiers
     source          1..3052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     terminator      complement(340..371)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(388..1065)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1072..1083
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1101..1119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1126..1144
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1303..1891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1383..1402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2062..2853,2854..2922))
                     /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     2685..2704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2923..3027)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc cgagaccgta gtcaaaagcc tccggtcgga
      361 ggcttttgac ttggctgagg agtgccatta agcaccggtg gagtgacgac cttcagcacg
      421 ttcgtactgt tcaacgatgg tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc
      481 ggttttgtaa gcacccggca gctgaaccgg ttttttagcc atgtaggtgg ttttaacttc
      541 agcgtcgtag tgaccaccgt ctttcagttt cagacgcatt ttgatttcac ctttcagagc
      601 accgtcttcc gggtacatac gttcggtgga agcttcccaa cccatggttt ttttctgcat
      661 aaccggaccg tcggacggga agttggtacc acgcagttta actttgtaga tgaactcacc
      721 gtcttgcagg gaggagtcct gggtaacggt aacaacacca ccgtcttcga agttcataac
      781 acgttcccat ttgaaacctt ccgggaagga cagtttcagg tagtccggga tgtcagccgg
      841 gtgtttaacg taagctttgg aaccgtactg gaactgcggg gacaggatgt cccaagcgaa
      901 cggcagcgga ccacctttgg taactttcag tttagcggtc tgggtacctt cgtacggacg
      961 accttcacct tcaccttcga tttcgaactc gtgaccgtta acggaacctt ccatacgaac
     1021 tttgaaacgc atgaactctt tgataacgtc ttcgctactc gccatagatc atttctcctc
     1081 tttcagatcc gtgctcagta tctctatcac tgatagggat gtcaatctct atcactgata
     1141 gggaggtctc gtcaggagcc ccccccctga acccaggata accctcaaag tcggggggct
     1201 ttttttaagc ttgggccgct cgaggtacct ctctacatat gacatgtgag caaaaggcca
     1261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1921 tgacgctcag tggaacgaaa acttacgtta agggattttg gtcatgagat tatcaaaaag
     1981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagatccac gctcaccggc
     2221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ct
//
