LOCUS       sequence-422188-        3223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using U6-3' MS2-CircRNA in 
            HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3223)
  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 3223)
  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: U6-3' MS2-CircRNA
FEATURES             Location/Qualifiers
     source          1..3223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        474..722
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     474..494
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     645..664
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        829..847
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1002..1018)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1002..1018)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1015..1037)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1026..1042
                     /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(1050..1056,1057..1074,1075..1080))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1095..1116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1233..1250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1404..1992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1484..1503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2163..2954,2955..3023))
                     /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     2786..2805
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3024..3128)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3196..3214
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accagatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac
      421 gccaagcttg catgcctgca ggtcgcccta ggggtaccaa ggtcgggcag gaagagggcc
      481 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
      541 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
      601 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
      661 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
      721 ccggccatca gtcgccggtc ccaagcccgg ataaaatggg agggggcggg aaaccgccta
      781 accatgccga ctgatggcag aactaacgag accggatccg gtctctgcac atgaggatca
      841 cccatgtgca aattaacact gccatcagtc ggcgtggact gtagaacact gccaatgccg
      901 gtcccaagcc cggataaaag tggagggtac agtccacgct tttttttgaa ttcgtaatca
      961 tggttccatc ccaatggcgc gccgagcttg gcgtaatcat ggtcatagct gtttcctgtg
     1021 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     1081 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1141 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1201 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1801 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     1861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2041 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2101 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2161 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2221 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2281 cccagtgctg caatgatacc gcgactccca cgctcaccgg ctccagattt atcagcaata
     2341 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2401 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2461 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2521 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2581 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2641 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2701 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2761 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     2821 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     2881 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     2941 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3001 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3061 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3121 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3181 acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
