LOCUS       sequence-364379-        3004 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 3004)
  AUTHORS   Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, 
            Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR
  TITLE     Search-and-replace genome editing without double-strand breaks or 
            donor DNA.
  JOURNAL   Nature. 2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 
            10.1038/s41586-019-1711-4.
  PUBMED    31634902
REFERENCE   2  (bases 1 to 3004)
  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: "Nature. 
            2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 
            10.1038/s41586-019-1711-4."
COMMENT     Sequence Label: pU6-pegRNA-GG-acceptor
FEATURES             Location/Qualifiers
     source          1..3004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    502..520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    527..545
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             563..574
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             581..1258
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1275..1306
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        complement(1316..1564)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1374..1393)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1544..1564)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1671..1775
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1776..1844,1845..2636)
                     /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     complement(1994..2013)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2807..3004,1..391)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
       61 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      121 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
      181 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
      241 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      301 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      361 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      421 cctggccttt tgctggcctt ttgctcacat gtcatatgta gagaggtacc tcgagcggcc
      481 caagcttaaa aaaacgagac ctccctatca gtgatagaga ttgacatccc tatcagtgat
      541 agagatactg agcacggatc tgaaagagga gaaaggatct atggcgagta gcgaagacgt
      601 tatcaaagag ttcatgcgtt tcaaagttcg tatggaaggt tccgttaacg gtcacgagtt
      661 cgaaatcgaa ggtgaaggtg aaggtcgtcc gtacgaaggt acccagaccg ctaaactgaa
      721 agttaccaaa ggtggtccgc tgccgttcgc ttgggacatc ctgtccccgc agttccagta
      781 cggttccaaa gcttacgtta aacacccggc tgacatcccg gactacctga aactgtcctt
      841 cccggaaggt ttcaaatggg aacgtgttat gaacttcgaa gacggtggtg ttgttaccgt
      901 tacccaggac tcctccctgc aagacggtga gttcatctac aaagttaaac tgcgtggtac
      961 caacttcccg tccgacggtc cggttatgca gaaaaaaacc atgggttggg aagcttccac
     1021 cgaacgtatg tacccggaag acggtgctct gaaaggtgaa atcaaaatgc gtctgaaact
     1081 gaaagacggt ggtcactacg acgctgaagt taaaaccacc tacatggcta aaaaaccggt
     1141 tcagctgccg ggtgcttaca aaaccgacat caaactggac atcacctccc acaacgaaga
     1201 ctacaccatc gttgaacagt acgaacgtgc tgaaggtcgt cactccaccg gtgcttaatg
     1261 gcactcctca gccaagtcaa aagcctccga ccggaggctt ttgactacgg tctcgggtgt
     1321 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     1381 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     1441 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa
     1501 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     1561 cctcttcctg cccgaccttg gtaccggatc cagtcgactg aattggttcc tttaaagcct
     1621 gcttttttgt acagctagcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1681 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1741 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1801 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1861 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1921 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1981 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2041 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     2101 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     2161 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     2221 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     2281 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     2341 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2401 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2461 ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg tatcattgca gcactggggc
     2521 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2581 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2641 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2701 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2761 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2821 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2881 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2941 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3001 cacc
//
