LOCUS       sequence-422195-        3225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using U6-5' MS2-CircRNA in 
            HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3225)
  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 3225)
  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: U6-5' MS2-CircRNA
FEATURES             Location/Qualifiers
     source          1..3225
                     /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        809..827
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1004..1020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1004..1020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1017..1039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1028..1044
                     /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(1052..1058,1059..1076,1077..1082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1097..1118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1235..1252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1406..1994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1486..1505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2165..2956,2957..3025))
                     /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     2788..2807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3026..3130)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3198..3216
                     /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 aactaagcac atgaggatca cccatgtgcc gagaccttcc
      841 gataggtctc taaattaaca ctgccatcag tcggcgtgga ctgtagaaca ctgccaatgc
      901 cggtcccaag cccggataaa agtggagggt acagtccacg cttttttttg aattcgtaat
      961 catggttcca tcccaatggc gcgccgagct tggcgtaatc atggtcatag ctgtttcctg
     1021 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1081 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1141 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1201 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2281 gccccagtgc tgcaatgata ccgcgactcc cacgctcacc ggctccagat ttatcagcaa
     2341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3181 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
