LOCUS       sequence-422200-        3060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using LbCas12a-crRNA in 
            HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3060)
  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 3060)
  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: LbCas12a-crRNA
FEATURES             Location/Qualifiers
     source          1..3060
                     /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"
     primer_bind     complement(839..855)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(839..855)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(852..874)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    863..879
                     /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(887..893,894..911,912..917))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    932..953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1070..1087)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1241..1829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1321..1340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2000..2791,2792..2860))
                     /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     2623..2642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2861..2965)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3033..3051
                     /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 ccgtaatttc tactaagtgt agattgagac cctgcagggt ctcttttttt ttgagctcaa
      781 gcttgaattc gtaatcatgg ttccatccca atggcgcgcc gagcttggcg taatcatggt
      841 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      901 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      961 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1021 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1081 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1141 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1201 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1261 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1321 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1381 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1441 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1501 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1561 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1621 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1681 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1741 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1801 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1861 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1921 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     1981 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2041 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2101 agggcttacc atctggcccc agtgctgcaa tgataccgcg actcccacgc tcaccggctc
     2161 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2221 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2281 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2341 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2401 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2461 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2521 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2581 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2641 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2701 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     2761 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2821 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2881 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2941 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     3001 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
