LOCUS       sequence-422189-        3252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using U6-5'+3' MS2-CircRNA in
            HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3252)
  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 3252)
  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'+3' MS2-CircRNA
FEATURES             Location/Qualifiers
     source          1..3252
                     /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"
     misc_RNA        858..876
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1031..1047)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1031..1047)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1044..1066)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1055..1071
                     /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(1079..1085,1086..1103,1104..1109))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1124..1145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1262..1279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1433..2021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1513..1532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2192..2983,2984..3052))
                     /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     2815..2834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3053..3157)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3225..3243
                     /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 tcaaggcaca tgaggatcac ccatgtgcaa attaacactg ccatcagtcg
      901 gcgtggactg tagaacactg ccaatgccgg tcccaagccc ggataaaagt ggagggtaca
      961 gtccacgctt ttttttgaat tcgtaatcat ggttccatcc caatggcgcg ccgagcttgg
     1021 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1081 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1141 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1201 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1261 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     1321 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1381 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1441 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1501 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1561 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1621 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1681 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1741 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1801 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1861 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1921 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1981 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2041 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2101 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2161 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2221 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     2281 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgactcccac
     2341 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     2401 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     2461 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     2521 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     2581 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2641 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2701 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2761 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2821 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2881 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2941 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3001 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3061 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3121 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3181 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     3241 ggccctttcg tc
//
