LOCUS       sequence-422202-        7209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For circular RNA-mediated prime editor using nLbCas12a-D156R-R1138A 
            in HEK293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7209)
  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 7209)
  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: nLbCas12a
FEATURES             Location/Qualifiers
     source          1..7209
                     /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             721..4401
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFRNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMANSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VK"
     CDS             4444..4464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4473..4490
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4513..4530)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4519..4743
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4814..4830)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4814..4830)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4827..4849)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4838..4854
                     /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(4862..4868,4869..4886,4887..4892))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4907..4928
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5045..5062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5216..5804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5296..5315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5975..6766,6767..6835))
                     /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     6598..6617
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6836..6940)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7019..7038)
                     /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 atgagcaagc tggagaagtt tacaaactgc tactccctgt ctaagaccct gaggttcaag
      781 gccatccctg tgggcaagac ccaggagaac atcgacaata agcggctgct ggtggaggac
      841 gagaagagag ccgaggatta taagggcgtg aagaagctgc tggatcgcta ctatctgtct
      901 tttatcaacg acgtgctgca cagcatcaag ctgaagaatc tgaacaatta catcagcctg
      961 ttccggaaga aaaccagaac cgagaaggag aataaggagc tggagaacct ggagatcaat
     1021 ctgcggaagg agatcgccaa ggccttcaag ggcaacgagg gctacaagtc cctgtttaag
     1081 aaggatatca tcgagacaat cctgccagag ttcctggacg ataaggacga gatcgccctg
     1141 gtgaacagct tcaatggctt taccacagcc ttcaccggct tctttcgtaa cagagagaat
     1201 atgttttccg aggaggccaa gagcacatcc atcgccttca ggtgtatcaa cgagaatctg
     1261 acccgctaca tctctaatat ggacatcttc gagaaggtgg acgccatctt tgataagcac
     1321 gaggtgcagg agatcaagga gaagatcctg aacagcgact atgatgtgga ggatttcttt
     1381 gagggcgagt tctttaactt tgtgctgaca caggagggca tcgacgtgta taacgccatc
     1441 atcggcggct tcgtgaccga gagcggcgag aagatcaagg gcctgaacga gtacatcaac
     1501 ctgtataatc agaaaaccaa gcagaagctg cctaagttta agccactgta taagcaggtg
     1561 ctgagcgatc gggagtctct gagcttctac ggcgagggct atacatccga tgaggaggtg
     1621 ctggaggtgt ttagaaacac cctgaacaag aacagcgaga tcttcagctc catcaagaag
     1681 ctggagaagc tgttcaagaa ttttgacgag tactctagcg ccggcatctt tgtgaagaac
     1741 ggccccgcca tcagcacaat ctccaaggat atcttcggcg agtggaacgt gatccgggac
     1801 aagtggaatg ccgagtatga cgatatccac ctgaagaaga aggccgtggt gaccgagaag
     1861 tacgaggacg atcggagaaa gtccttcaag aagatcggct ccttttctct ggagcagctg
     1921 caggagtacg ccgacgccga tctgtctgtg gtggagaagc tgaaggagat catcatccag
     1981 aaggtggatg agatctacaa ggtgtatggc tcctctgaga agctgttcga cgccgatttt
     2041 gtgctggaga agagcctgaa gaagaacgac gccgtggtgg ccatcatgaa ggacctgctg
     2101 gattctgtga agagcttcga gaattacatc aaggccttct ttggcgaggg caaggagaca
     2161 aacagggacg agtccttcta tggcgatttt gtgctggcct acgacatcct gctgaaggtg
     2221 gaccacatct acgatgccat ccgcaattat gtgacccaga agccctactc taaggataag
     2281 ttcaagctgt attttcagaa ccctcagttc atgggcggct gggacaagga taaggagaca
     2341 gactatcggg ccaccatcct gagatacggc tccaagtact atctggccat catggataag
     2401 aagtacgcca agtgcctgca gaagatcgac aaggacgatg tgaacggcaa ttacgagaag
     2461 atcaactata agctgctgcc cggccctaat aagatgctgc caaaggtgtt cttttctaag
     2521 aagtggatgg cctactataa ccccagcgag gacatccaga agatctacaa gaatggcaca
     2581 ttcaagaagg gcgatatgtt taacctgaat gactgtcaca agctgatcga cttctttaag
     2641 gatagcatct cccggtatcc aaagtggtcc aatgcctacg atttcaactt ttctgagaca
     2701 gagaagtata aggacatcgc cggcttttac agagaggtgg aggagcaggg ctataaggtg
     2761 agcttcgagt ctgccagcaa gaaggaggtg gataagctgg tggaggaggg caagctgtat
     2821 atgttccaga tctataacaa ggacttttcc gataagtctc acggcacacc caatctgcac
     2881 accatgtact tcaagctgct gtttgacgag aacaatcacg gacagatcag gctgagcgga
     2941 ggagcagagc tgttcatgag gcgcgcctcc ctgaagaagg aggagctggt ggtgcaccca
     3001 gccaactccc ctatcgccaa caagaatcca gataatccca agaaaaccac aaccctgtcc
     3061 tacgacgtgt ataaggataa gaggttttct gaggaccagt acgagctgca catcccaatc
     3121 gccatcaata agtgccccaa gaacatcttc aagatcaata cagaggtgcg cgtgctgctg
     3181 aagcacgacg ataaccccta tgtgatcggc atcgacaggg gcgagcgcaa tctgctgtat
     3241 atcgtggtgg tggacggcaa gggcaacatc gtggagcagt attccctgaa cgagatcatc
     3301 aacaacttca acggcatcag gatcaagaca gattaccact ctctgctgga caagaaggag
     3361 aaggagaggt tcgaggcccg ccagaactgg acctccatcg agaatatcaa ggagctgaag
     3421 gccggctata tctctcaggt ggtgcacaag atctgcgagc tggtggagaa gtacgatgcc
     3481 gtgatcgccc tggaggacct gaactctggc tttaagaata gccgcgtgaa ggtggagaag
     3541 caggtgtatc agaagttcga gaagatgctg atcgataagc tgaactacat ggtggacaag
     3601 aagtctaatc cttgtgcaac aggcggcgcc ctgaagggct atcagatcac caataagttc
     3661 gagagcttta agtccatgtc tacccagaac ggcttcatct tttacatccc tgcctggctg
     3721 acatccaaga tcgatccatc taccggcttt gtgaacctgc tgaaaaccaa gtataccagc
     3781 atcgccgatt ccaagaagtt catcagctcc tttgacagga tcatgtacgt gcccgaggag
     3841 gatctgttcg agtttgccct ggactataag aacttctctc gcacagacgc cgattacatc
     3901 aagaagtgga agctgtactc ctacggcaac cggatcagaa tcttccggaa tcctaagaag
     3961 aacaacgtgt tcgactggga ggaggtgtgc ctgaccagcg cctataagga gctgttcaac
     4021 aagtacggca tcaattatca gcagggcgat atcagagccc tgctgtgcga gcagtccgac
     4081 aaggccttct actctagctt tatggccctg atgagcctga tgctgcagat ggcgaacagc
     4141 atcacaggcc gcaccgacgt ggattttctg atcagccctg tgaagaactc cgacggcatc
     4201 ttctacgata gccggaacta tgaggcccag gagaatgcca tcctgccaaa gaacgccgac
     4261 gccaatggcg cctataacat cgccagaaag gtgctgtggg ccatcggcca gttcaagaag
     4321 gccgaggacg agaagctgga taaggtgaag atcgccatct ctaacaagga gtggctggag
     4381 tacgcccaga ccagcgtgaa gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc
     4441 gagcccaaga agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa
     4501 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4561 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgaga
     4621 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4681 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     4741 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     4801 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4861 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     4921 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4981 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5041 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5101 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5161 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5221 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5281 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5341 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5401 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5461 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5521 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5581 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5641 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5701 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5761 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5821 tttctacggg gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5881 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5941 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6001 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6061 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6121 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6181 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6241 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6301 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6361 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6421 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6481 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6541 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6601 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6661 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6721 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6781 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6841 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6901 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6961 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtct actctcagta
     7021 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7081 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     7141 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     7201 atacgcgtt
//
