LOCUS       sequence-400574-        7915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hSyn-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 7915)
  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 7915)
  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-hSyn-evoCjCas9-ABE8e_gRNA
FEATURES             Location/Qualifiers
     source          1..7915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        147..594
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             647..667
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4253..4273
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4287..4511
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4531..4603)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(4727..4975)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4785..4804)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4955..4975)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4976..5105
                     /label=AAV2 ITR
     primer_bind     complement(5124..5141)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5124..5140)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5133..5155)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5282..5737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5369..5388)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5579..5600
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5763..5867
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5868..5936,5937..6728)
                     /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(6086..6105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6899..7487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7388..7407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7775..7796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7811..7816,7817..7834,7835..7841)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7849..7865
                     /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     7854..7876
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7873..7889
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7873..7889
                     /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 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg
      181 ggtgggggtg cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt
      241 ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg
      301 tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc
      361 caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc
      421 cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg
      481 aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg
      541 ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga
      601 ggccgccacc atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg
      661 gaaagtctct gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc
      721 caagagggca cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag
      781 agtgatcggc gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga
      841 aattatggcc ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac
      901 cctgtacgtg acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat
      961 cggccgcgtg gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa
     1021 cgtgctgaac taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga
     1081 tgaatgtgcc gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca
     1141 gaagaaggcc cagagctcca tcaactccgg aggatctagc ggaggctcct ctggctctga
     1201 gacacctggc acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc
     1261 agcccgcatc ctggcctttg ccatcggcat cagcagcatc ggatgggcct tcagcgagaa
     1321 tgacgagctg aaggactgcg gtgtgagaat ctttacaaag gtcgagaacc ccaagaccgg
     1381 cgaaagcctg gcgctgccaa gacggctggc ccggtctgct cggaagagat acgcccggag
     1441 aaaagccaga ctgaatcacc tgaaacacct gatcgccaac gagttcaaac tgaactacga
     1501 ggactaccag agcttcgatg agagcttggc taaggcctac aaaggcagcc tgatcagccc
     1561 ctacgagctg agattcagag ctctgaatga gctcctgagc aagcaggact tcgctagagt
     1621 gatcctgcac atcgccaagc gaagaggcta cgacgacatc aagaactctg atgacaagga
     1681 gaagggcgcc attctgaaag ccatcaagca gaacgaagag aaactggcca attaccagag
     1741 cgtgggcgag tacctgtaca aggagtactt ccagaagttc aaagaaaata gcaaagagtt
     1801 caccaacgtg cggaacaaga aggagtctta tgaaagatgt atcgcccaga gcttcctgaa
     1861 agacgagctg aaactgatct tcaagaagca aagagagttt ggcttcagct tcagcaaaaa
     1921 atttgaagaa gaggtgctgt ctgtcgcctt ctacaagagg gctctgaagg acttcagcca
     1981 cctggtgggc aattgcagct ttttcacaga cgaaaagcgg gcccctaaga acagccctct
     2041 ggccttcatg ttcgtggctc tgaccagaat catcaacctg ctgaacaacc tgaaaaatac
     2101 cgagggcatc ctctatacca aggacgatct gaacgccctg ctgaatgagg tgctcaagaa
     2161 tggcaccctg acctacaagc aaacaaaaaa actgctgggc ctgtctgacg actacgagtt
     2221 taaaggcgag aagggcacct atttcatcga atttaagaag tacaaggaat tcatcaaggc
     2281 actgggcgaa cacaacctgt ctcaggacga cctgaacgag atcgccaagg acatcaccct
     2341 gatcaaggat gagatcaagc tgaaaaaagc tctggccaag tacgacctga atcaaaacca
     2401 gatcgacagc ttaagcaagc tggaatttaa ggatcacctg aacatctcct ttaaggccct
     2461 gaagctggtg accccactga tgctggaagg aaagaagtac gacgaagcat gcaacgagct
     2521 taacctgaag gttgctatca acgaggataa gaaggatttc ctgcctgcct ttaacgagac
     2581 atactacaag gatgaggtga ccaaccccgt ggtgctgaga gctatcaaag agtacagaaa
     2641 ggtgctgaac gccctgctga agaagtacgg caaggtccac aagatcaata ttgagctggc
     2701 ccgggaggtt ggaaagaacc actctcagag agcaaagatc gagaaggagc aaaacgagaa
     2761 ctacaaagcg aagaaggacg ccgaactgga gtgcgagaag cttggcctga agatcaactc
     2821 taagaatatc ctgaaactca gacttttcaa agaacagaag gaattctgtg cctacagcgg
     2881 cgagaagatc aaaatttctg acctgcagga tgaaaagatg ctggagatcg accacatcta
     2941 cccttactcc agaagcttcg acgacagcta tatgaacaaa gtgctggtgt tcacaaagca
     3001 gaaccaggag aagctgaatc agaccccttt cgaggccttc ggcaatgact ccgccaagtg
     3061 gcagaaaatc gaggtgctgg ccaaaaacct gccaaccaag aaacagaaga gaatcctcga
     3121 caagaactac aaggacaagg aacagaagaa cttcaaggat cggaacctga acgacacccg
     3181 gtacatcgcc aggctggtgt taaattacac caaggactac ctggatttcc tgcccctgag
     3241 cgacgacgag aacaccaagc tgaacgacac acagaagggc agcaaggtgc acgtggaagc
     3301 caagagcggc atgctgacca gcgcactgcg ccacacttgg ggcttcagcg ctaaggaccg
     3361 gaacaaccac ctgcatcacg ccatcgatgc cgtgatcata gcctacgcca acaactcaat
     3421 cgtgaaagct ttcagtgact ttaagaaaga acaggagagc aactctgccg aactgtacgc
     3481 caagaaaatt agcgagctgg actacaagaa caaacggaag ttcttcgaac ctttctcagg
     3541 atttagacag aaggtgctgg ataagatcga tgaaatcttc gtgtccaagc ccgagagaaa
     3601 gaagcctagc ggagccctgc acaaggaaac cttcagaaag gaagaagagt tctaccagtc
     3661 ttatgggggc aaagaaggcg tgctgaaggc cctggaactc ggcaagatca gaaaggtgaa
     3721 gggaaagatt gtgaagaacg gcgacatgtt cagagtggac atcttcaagc acaagaagac
     3781 caacaagttc tatgccgtgc ctatctacac aatggatttc gccctgaaag tgctgcctaa
     3841 caaggctgtc gccagatcta agaagggcga gatcaaggac tggatcctga tggacgaaaa
     3901 ctacgagttc tgcttcagcc tgtacaagga cagcctgatc ctgatccaaa caaagaagat
     3961 gcaggagcct gaattcgtgt actacaacgc cttcacaagc agcaccgtga gcctgatcgt
     4021 gtctaaacat gataacaagt tcgaaaccct gtccaagaac caaaagatcc tgttcaagaa
     4081 cgccaacgag aaggaagtga tcgccaaggg catcggcatt cagaatctga aggtgttcga
     4141 gaaatatatc gtgtccgctc tgggagaggt tacaaaggcc gagtttcggc agagagaaga
     4201 ttttaagaag tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa
     4261 gaagaggaaa gtctaataga tctcgactgt gccttctagt tgccagccat ctgttgtttg
     4321 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4381 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4441 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4501 gggctctatg gctcgagcgg cccaaaaaaa gcggttttag gggattgtaa ccccgcagag
     4561 tcccgcaaac tctttattct agtccctttt cagggactag aacagagacg tacaaaaaag
     4621 agcaagaagc taaaaaagat ttaaaaatta tttttagcgc agttaatgga acaggaacta
     4681 aatttacccc aaaatgtatt ttgtgtgctc atttacaacc gtctccggtg tttcgtcctt
     4741 tccacaagat atataaagcc aagaaatcga aatactttca agttacggta agcatatgat
     4801 agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac
     4861 gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcta attatctctc
     4921 taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcaggaa
     4981 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     5041 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     5101 cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     5161 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5221 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5281 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5341 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5401 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5461 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5521 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5581 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5641 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5701 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     5761 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5821 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5881 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5941 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6001 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6061 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6121 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6181 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6241 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6301 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6361 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6421 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6481 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6541 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6601 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6661 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6721 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6781 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6841 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6901 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6961 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7021 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7081 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7141 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7201 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7261 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7321 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7381 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7441 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7501 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7561 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7621 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     7681 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     7741 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     7801 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     7861 aacaatttca cacaggaaac agctatgacc atgattacgc cagatttaat taagg
//
