LOCUS       sequence-400578-        7898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P3-evoCjCas9-ABE8e and hU6-BsmBI-gRNA in AAV2 backbone (ITRs).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7898)
  AUTHORS   Schmidheini L, Mathis N, Marquart KF, Rothgangl T, Kissling L, Bock 
            D, Chanez C, Wang JP, Jinek M, Schwank G
  TITLE     Continuous directed evolution of a compact CjCas9 variant with broad
            PAM compatibility.
  JOURNAL   Nat Chem Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x.
  PUBMED    37735239
REFERENCE   2  (bases 1 to 7898)
  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 Chem 
            Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x."
COMMENT     Sequence Label: AAV-P3-evoCjCas9-ABE8e_gRNA
FEATURES             Location/Qualifiers
     source          1..7898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     intron          451..583
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             630..650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4236..4256
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4270..4494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4514..4586)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(4710..4958)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4768..4787)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4938..4958)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4959..5088
                     /label=AAV2 ITR
     primer_bind     complement(5107..5124)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5107..5123)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5116..5138)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5265..5720
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5352..5371)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5562..5583
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5746..5850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5851..5919,5920..6711)
                     /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     complement(6069..6088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6882..7470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7371..7390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7758..7779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7794..7799,7800..7817,7818..7824)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7832..7848
                     /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)."
     primer_bind     7837..7859
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7856..7872
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7856..7872
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct accggcgcgc cgggggaggc tgctggtgaa tattaaccaa ggtcacccca
      181 gttatcggag gagcaaacag gggctaagtc cacacgcgtg gtaccgtctg tctgcacatt
      241 tcgtagagcg agtgttccga tactctaatc tccctaggca aggttcatat ttgtgtaggt
      301 tacttattct ccttttgttg actaagtcaa taatcagaat cagcaggttt ggagtcagct
      361 tggcagggat cagcagcctg ggttggaagg agggggtata aaagcccctt caccaggaga
      421 agccgtcaca cagatccaca agctcctggg gtaagtatca aggttacaag acaggtttaa
      481 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
      541 cctattggtc ttactgacat ccactttgcc tttctctcca cagggccgcc accatgaaac
      601 ggacagccga cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc tctgaggtgg
      661 agttttccca cgagtactgg atgagacatg ccctgaccct ggccaagagg gcacgggatg
      721 agagggaggt gcctgtggga gccgtgctgg tgctgaacaa tagagtgatc ggcgagggct
      781 ggaacagagc catcggcctg cacgacccaa cagcccatgc cgaaattatg gccctgagac
      841 agggcggcct ggtcatgcag aactacagac tgattgacgc caccctgtac gtgacattcg
      901 agccttgcgt gatgtgcgcc ggcgccatga tccactctag gatcggccgc gtggtgtttg
      961 gcgtgaggaa ctcaaaaaga ggcgccgcag gctccctgat gaacgtgctg aactaccccg
     1021 gcatgaatca ccgcgtcgaa attaccgagg gaatcctggc agatgaatgt gccgccctgc
     1081 tgtgcgattt ctatcggatg cctagacagg tgttcaatgc tcagaagaag gcccagagct
     1141 ccatcaactc cggaggatct agcggaggct cctctggctc tgagacacct ggcacaagcg
     1201 agagcgcaac acctgaaagc agcgggggca gcagcggggg gtcagcccgc atcctggcct
     1261 ttgccatcgg catcagcagc atcggatggg ccttcagcga gaatgacgag ctgaaggact
     1321 gcggtgtgag aatctttaca aaggtcgaga accccaagac cggcgaaagc ctggcgctgc
     1381 caagacggct ggcccggtct gctcggaaga gatacgcccg gagaaaagcc agactgaatc
     1441 acctgaaaca cctgatcgcc aacgagttca aactgaacta cgaggactac cagagcttcg
     1501 atgagagctt ggctaaggcc tacaaaggca gcctgatcag cccctacgag ctgagattca
     1561 gagctctgaa tgagctcctg agcaagcagg acttcgctag agtgatcctg cacatcgcca
     1621 agcgaagagg ctacgacgac atcaagaact ctgatgacaa ggagaagggc gccattctga
     1681 aagccatcaa gcagaacgaa gagaaactgg ccaattacca gagcgtgggc gagtacctgt
     1741 acaaggagta cttccagaag ttcaaagaaa atagcaaaga gttcaccaac gtgcggaaca
     1801 agaaggagtc ttatgaaaga tgtatcgccc agagcttcct gaaagacgag ctgaaactga
     1861 tcttcaagaa gcaaagagag tttggcttca gcttcagcaa aaaatttgaa gaagaggtgc
     1921 tgtctgtcgc cttctacaag agggctctga aggacttcag ccacctggtg ggcaattgca
     1981 gctttttcac agacgaaaag cgggccccta agaacagccc tctggccttc atgttcgtgg
     2041 ctctgaccag aatcatcaac ctgctgaaca acctgaaaaa taccgagggc atcctctata
     2101 ccaaggacga tctgaacgcc ctgctgaatg aggtgctcaa gaatggcacc ctgacctaca
     2161 agcaaacaaa aaaactgctg ggcctgtctg acgactacga gtttaaaggc gagaagggca
     2221 cctatttcat cgaatttaag aagtacaagg aattcatcaa ggcactgggc gaacacaacc
     2281 tgtctcagga cgacctgaac gagatcgcca aggacatcac cctgatcaag gatgagatca
     2341 agctgaaaaa agctctggcc aagtacgacc tgaatcaaaa ccagatcgac agcttaagca
     2401 agctggaatt taaggatcac ctgaacatct cctttaaggc cctgaagctg gtgaccccac
     2461 tgatgctgga aggaaagaag tacgacgaag catgcaacga gcttaacctg aaggttgcta
     2521 tcaacgagga taagaaggat ttcctgcctg cctttaacga gacatactac aaggatgagg
     2581 tgaccaaccc cgtggtgctg agagctatca aagagtacag aaaggtgctg aacgccctgc
     2641 tgaagaagta cggcaaggtc cacaagatca atattgagct ggcccgggag gttggaaaga
     2701 accactctca gagagcaaag atcgagaagg agcaaaacga gaactacaaa gcgaagaagg
     2761 acgccgaact ggagtgcgag aagcttggcc tgaagatcaa ctctaagaat atcctgaaac
     2821 tcagactttt caaagaacag aaggaattct gtgcctacag cggcgagaag atcaaaattt
     2881 ctgacctgca ggatgaaaag atgctggaga tcgaccacat ctacccttac tccagaagct
     2941 tcgacgacag ctatatgaac aaagtgctgg tgttcacaaa gcagaaccag gagaagctga
     3001 atcagacccc tttcgaggcc ttcggcaatg actccgccaa gtggcagaaa atcgaggtgc
     3061 tggccaaaaa cctgccaacc aagaaacaga agagaatcct cgacaagaac tacaaggaca
     3121 aggaacagaa gaacttcaag gatcggaacc tgaacgacac ccggtacatc gccaggctgg
     3181 tgttaaatta caccaaggac tacctggatt tcctgcccct gagcgacgac gagaacacca
     3241 agctgaacga cacacagaag ggcagcaagg tgcacgtgga agccaagagc ggcatgctga
     3301 ccagcgcact gcgccacact tggggcttca gcgctaagga ccggaacaac cacctgcatc
     3361 acgccatcga tgccgtgatc atagcctacg ccaacaactc aatcgtgaaa gctttcagtg
     3421 actttaagaa agaacaggag agcaactctg ccgaactgta cgccaagaaa attagcgagc
     3481 tggactacaa gaacaaacgg aagttcttcg aacctttctc aggatttaga cagaaggtgc
     3541 tggataagat cgatgaaatc ttcgtgtcca agcccgagag aaagaagcct agcggagccc
     3601 tgcacaagga aaccttcaga aaggaagaag agttctacca gtcttatggg ggcaaagaag
     3661 gcgtgctgaa ggccctggaa ctcggcaaga tcagaaaggt gaagggaaag attgtgaaga
     3721 acggcgacat gttcagagtg gacatcttca agcacaagaa gaccaacaag ttctatgccg
     3781 tgcctatcta cacaatggat ttcgccctga aagtgctgcc taacaaggct gtcgccagat
     3841 ctaagaaggg cgagatcaag gactggatcc tgatggacga aaactacgag ttctgcttca
     3901 gcctgtacaa ggacagcctg atcctgatcc aaacaaagaa gatgcaggag cctgaattcg
     3961 tgtactacaa cgccttcaca agcagcaccg tgagcctgat cgtgtctaaa catgataaca
     4021 agttcgaaac cctgtccaag aaccaaaaga tcctgttcaa gaacgccaac gagaaggaag
     4081 tgatcgccaa gggcatcggc attcagaatc tgaaggtgtt cgagaaatat atcgtgtccg
     4141 ctctgggaga ggttacaaag gccgagtttc ggcagagaga agattttaag aagtctggcg
     4201 gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaat
     4261 agatctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4321 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4381 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4441 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggctcgag
     4501 cggcccaaaa aaagcggttt taggggattg taaccccgca gagtcccgca aactctttat
     4561 tctagtccct tttcagggac tagaacagag acgtacaaaa aagagcaaga agctaaaaaa
     4621 gatttaaaaa ttatttttag cgcagttaat ggaacaggaa ctaaatttac cccaaaatgt
     4681 attttgtgtg ctcatttaca accgtctccg gtgtttcgtc ctttccacaa gatatataaa
     4741 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
     4801 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
     4861 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
     4921 atatgcaaat atgaaggaat catgggaaat aggccctcag gaacccctag tgatggagtt
     4981 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     5041 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc
     5101 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5161 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5221 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5281 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5341 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5401 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5461 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5521 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5581 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5641 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5701 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5761 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5821 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5881 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5941 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6001 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6061 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6121 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6181 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6241 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6301 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6361 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6421 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6481 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6541 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6601 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6661 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6721 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6781 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6841 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6901 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6961 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7021 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7081 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7141 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7201 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7261 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7321 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7381 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7441 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7501 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     7561 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     7621 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     7681 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7741 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     7801 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     7861 aacagctatg accatgatta cgccagattt aattaagg
//
