LOCUS       sequence-422192-        5541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using MCP-M-MLV RT∆RNase H in
            HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5541)
  AUTHORS   Liang R, He Z, Zhao KT, Zhu H, Hu J, Liu G, Gao Q, Liu M, Zhang R, 
            Qiu JL, Gao C
  TITLE     Prime editing using CRISPR-Cas12a and circular RNAs in human cells.
  JOURNAL   Nat Biotechnol. 2024 Jan 10. doi: 10.1038/s41587-023-02095-x.
  PUBMED    38200119
REFERENCE   2  (bases 1 to 5541)
  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. 2024 Jan 10. doi: 10.1038/s41587-023-02095-x."
COMMENT     Sequence Label: MCP-M-MLV RT∆RNase H
FEATURES             Location/Qualifiers
     source          1..5541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             694..714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2776..2796
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2805..2822
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2845..2862)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2851..3075
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3146..3162)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3146..3162)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3159..3181)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3170..3186
                     /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(3194..3200,3201..3218,3219..3224))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3239..3260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3377..3394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3548..4136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3628..3647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4307..5098,5099..5167))
                     /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     4930..4949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5168..5272)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5351..5370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcaagctt
      721 gcgtccaact tcacccaatt tgtactggtg gacaatggtg gaacaggaga cgtgaccgtg
      781 gccccttcta acttcgccaa tggaatcgca gagtggattt cctcaaactc ccggtcacag
      841 gcatacaaag tgacgtgttc agtgaggcag tcctccgctc agaaccgcaa atacaccatt
      901 aaggttgaag tgccaaaagg cgcctggagg tcctacttga atatggagct taccattcca
      961 atttttgcca caaatagcga ttgtgagctg attgtgaagg ccatgcaggg cctcctgaag
     1021 gacgggaacc ccattccgag cgccatagca gcaaatagcg gaatttattc tagactgcag
     1081 tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag cgagtcagca
     1141 acaccagaga gcagtggcgg cagcagcggc ggcagcgagc tccccgggag caccctaaat
     1201 atagaagatg agtatcggct acatgagacc tcaaaagagc cagatgtttc tctagggtcc
     1261 acatggctgt ctgattttcc tcaggcctgg gcggaaaccg ggggcatggg actggcagtt
     1321 cgccaagctc ctctgatcat acctctgaaa gcaacctcta cccccgtgtc cataaaacaa
     1381 taccccatgt cacaagaagc cagactgggg atcaagcccc acatacagag actgttggac
     1441 cagggaatac tggtaccctg ccagtccccc tggaacacgc ccctgctacc cgttaagaaa
     1501 ccagggacta atgattatag gcctgtccag gatctgagag aagtcaacaa gcgggtggaa
     1561 gacatccacc ccaccgtgcc caacccttac aacctcttga gcgggctccc accgtcccac
     1621 cagtggtaca ctgtgcttga tttaaaggat gcctttttct gcctgagact ccaccccacc
     1681 agtcagcctc tcttcgcctt tgagtggaga gatccagaga tgggaatctc aggacaattg
     1741 acctggacca gactcccaca gggtttcaaa aacagtccca ccctgtttaa tgaggcactg
     1801 cacagagacc tagcagactt ccggatccag cacccagact tgatcctgct acagtacgtg
     1861 gatgacttac tgctggccgc cacttctgag ctagactgcc aacaaggtac tcgggccctg
     1921 ttacaaaccc tagggaacct cgggtatcgg gcctcggcca agaaagccca aatttgccag
     1981 aaacaggtca agtatctggg gtatcttcta aaagagggtc agagatggct gactgaggcc
     2041 agaaaagaga ctgtgatggg gcagcctact ccgaagaccc ctcgacaact aagggagttc
     2101 ctagggaagg caggcttctg tcgcctcttc atccctgggt ttgcagaaat ggcagccccc
     2161 ctgtaccctc tcaccaaacc ggggactctg tttaattggg gcccagacca acaaaaggcc
     2221 tatcaagaaa tcaagcaagc tcttctaact gccccagccc tggggttgcc agatttgact
     2281 aagccctttg aactctttgt cgacgagaag cagggctacg ccaaaggtgt cctaacgcaa
     2341 aaactgggac cttggcgtcg gccggtggcc tacctgtcca aaaagctaga cccagtagca
     2401 gctgggtggc ccccttgcct acggatggta gcagccattg ccgtactgac aaaggatgca
     2461 ggcaagctaa ccatgggaca gccactagtc attctggccc cccatgcagt agaggcacta
     2521 gtcaaacaac cccccgaccg ctggctttcc aacgcccgga tgactcacta tcaggccttg
     2581 cttttggaca cggaccgggt ccagttcgga ccggtggtag ccctgaaccc ggctacgctg
     2641 ctcccactgc ctgaggaagg gctgcaacac aactgccttg atatcctggc cgaagcccac
     2701 ggaacccgac ccgacctaac ggaccagccg ctctctggcg gctcaaaaag aaccgccgac
     2761 ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac cggtcatcat caccatcacc
     2821 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2881 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2941 aataaaatga gaaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3001 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3061 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct
     3121 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3181 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
     3241 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3301 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3361 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3421 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3481 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3541 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3601 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3661 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3721 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3781 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3841 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3901 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3961 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4021 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4081 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4141 atcctttgat cttttctacg gggtctgaca ctcagtggaa cgaaaactca cgttaaggga
     4201 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4261 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4321 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4381 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4441 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4501 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4561 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4621 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4681 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4741 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4801 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4861 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4921 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4981 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5041 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5101 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5161 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5221 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5281 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     5341 ctactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     5401 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     5461 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     5521 tacgggccag atatacgcgt t
//
