LOCUS       sequence-409106-        8042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P3-evoCjCas9-TadCDd and hU6-BsmBI-gRNA in AAV2 backbone (ITRs).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8042)
  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 8042)
  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-TadCDd_gRNA
FEATURES             Location/Qualifiers
     source          1..8042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     CDS             495..515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4089..4337
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4380..4400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4414..4638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4658..4730)
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     promoter        complement(4854..5102)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4912..4931)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5082..5102)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   5103..5232
                     /label=AAV2 ITR
     primer_bind     complement(5251..5268)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5251..5267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5260..5282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5409..5864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5496..5515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5706..5727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5890..5994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5995..6063,6064..6855)
                     /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(6213..6232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7026..7614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7515..7534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7902..7923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7938..7943,7944..7961,7962..7968)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7976..7992
                     /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     7981..8003
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8000..8016
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8000..8016
                     /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 ccgccaccat gaaacggaca gccgacggaa
      481 gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt tcccacgagt
      541 actggatgag acatgccctg accctggcca agagggcacg ggatgagagg aaggcccctg
      601 tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac agagccatcg
      661 gcctgcacga cccaacagcc catgccgaaa ttatcgccct gagacagggc ggcctggtca
      721 tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct tgcgtgatgt
      781 gcgccggcgc catgatcaac tctaggatcg gccgcgtggt gtttggcgtg aggaactcaa
      841 aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg aatcaccgcg
      901 tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc gatttctatc
      961 ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc aactccggag
     1021 gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc gcaacacctg
     1081 aaagcagcgg gggcagcagc ggggggtcag cccgcatcct ggcctttgcc atcggcatca
     1141 gcagcatcgg atgggccttc agcgagaatg acgagctgaa ggactgcggt gtgagaatct
     1201 ttacaaaggt cgagaacccc aagaccggcg aaagcctggc gctgccaaga cggctggccc
     1261 ggtctgctcg gaagagatac gcccggagaa aagccagact gaatcacctg aaacacctga
     1321 tcgccaacga gttcaaactg aactacgagg actaccagag cttcgatgag agcttggcta
     1381 aggcctacaa aggcagcctg atcagcccct acgagctgag attcagagct ctgaatgagc
     1441 tcctgagcaa gcaggacttc gctagagtga tcctgcacat cgccaagcga agaggctacg
     1501 acgacatcaa gaactctgat gacaaggaga agggcgccat tctgaaagcc atcaagcaga
     1561 acgaagagaa actggccaat taccagagcg tgggcgagta cctgtacaag gagtacttcc
     1621 agaagttcaa agaaaatagc aaagagttca ccaacgtgcg gaacaagaag gagtcttatg
     1681 aaagatgtat cgcccagagc ttcctgaaag acgagctgaa actgatcttc aagaagcaaa
     1741 gagagtttgg cttcagcttc agcaaaaaat ttgaagaaga ggtgctgtct gtcgccttct
     1801 acaagagggc tctgaaggac ttcagccacc tggtgggcaa ttgcagcttt ttcacagacg
     1861 aaaagcgggc ccctaagaac agccctctgg ccttcatgtt cgtggctctg accagaatca
     1921 tcaacctgct gaacaacctg aaaaataccg agggcatcct ctataccaag gacgatctga
     1981 acgccctgct gaatgaggtg ctcaagaatg gcaccctgac ctacaagcaa acaaaaaaac
     2041 tgctgggcct gtctgacgac tacgagttta aaggcgagaa gggcacctat ttcatcgaat
     2101 ttaagaagta caaggaattc atcaaggcac tgggcgaaca caacctgtct caggacgacc
     2161 tgaacgagat cgccaaggac atcaccctga tcaaggatga gatcaagctg aaaaaagctc
     2221 tggccaagta cgacctgaat caaaaccaga tcgacagctt aagcaagctg gaatttaagg
     2281 atcacctgaa catctccttt aaggccctga agctggtgac cccactgatg ctggaaggaa
     2341 agaagtacga cgaagcatgc aacgagctta acctgaaggt tgctatcaac gaggataaga
     2401 aggatttcct gcctgccttt aacgagacat actacaagga tgaggtgacc aaccccgtgg
     2461 tgctgagagc tatcaaagag tacagaaagg tgctgaacgc cctgctgaag aagtacggca
     2521 aggtccacaa gatcaatatt gagctggccc gggaggttgg aaagaaccac tctcagagag
     2581 caaagatcga gaaggagcaa aacgagaact acaaagcgaa gaaggacgcc gaactggagt
     2641 gcgagaagct tggcctgaag atcaactcta agaatatcct gaaactcaga cttttcaaag
     2701 aacagaagga attctgtgcc tacagcggcg agaagatcaa aatttctgac ctgcaggatg
     2761 aaaagatgct ggagatcgac cacatctacc cttactccag aagcttcgac gacagctata
     2821 tgaacaaagt gctggtgttc acaaagcaga accaggagaa gctgaatcag acccctttcg
     2881 aggccttcgg caatgactcc gccaagtggc agaaaatcga ggtgctggcc aaaaacctgc
     2941 caaccaagaa acagaagaga atcctcgaca agaactacaa ggacaaggaa cagaagaact
     3001 tcaaggatcg gaacctgaac gacacccggt acatcgccag gctggtgtta aattacacca
     3061 aggactacct ggatttcctg cccctgagcg acgacgagaa caccaagctg aacgacacac
     3121 agaagggcag caaggtgcac gtggaagcca agagcggcat gctgaccagc gcactgcgcc
     3181 acacttgggg cttcagcgct aaggaccgga acaaccacct gcatcacgcc atcgatgccg
     3241 tgatcatagc ctacgccaac aactcaatcg tgaaagcttt cagtgacttt aagaaagaac
     3301 aggagagcaa ctctgccgaa ctgtacgcca agaaaattag cgagctggac tacaagaaca
     3361 aacggaagtt cttcgaacct ttctcaggat ttagacagaa ggtgctggat aagatcgatg
     3421 aaatcttcgt gtccaagccc gagagaaaga agcctagcgg agccctgcac aaggaaacct
     3481 tcagaaagga agaagagttc taccagtctt atgggggcaa agaaggcgtg ctgaaggccc
     3541 tggaactcgg caagatcaga aaggtgaagg gaaagattgt gaagaacggc gacatgttca
     3601 gagtggacat cttcaagcac aagaagacca acaagttcta tgccgtgcct atctacacaa
     3661 tggatttcgc cctgaaagtg ctgcctaaca aggctgtcgc cagatctaag aagggcgaga
     3721 tcaaggactg gatcctgatg gacgaaaact acgagttctg cttcagcctg tacaaggaca
     3781 gcctgatcct gatccaaaca aagaagatgc aggagcctga attcgtgtac tacaacgcct
     3841 tcacaagcag caccgtgagc ctgatcgtgt ctaaacatga taacaagttc gaaaccctgt
     3901 ccaagaacca aaagatcctg ttcaagaacg ccaacgagaa ggaagtgatc gccaagggca
     3961 tcggcattca gaatctgaag gtgttcgaga aatatatcgt gtccgctctg ggagaggtta
     4021 caaaggccga gtttcggcag agagaagatt ttaagaagag cggcgggagc ggcgggagcg
     4081 gggggagcac taatctgagc gacatcattg agaaggagac tgggaaacag ctggtcattc
     4141 aggagtccat cctgatgctg cctgaggagg tggaggaagt gatcggcaac aagccagagt
     4201 ctgacatcct ggtgcacacc gcctacgacg agtccacaga tgagaatgtg atgctgctga
     4261 cctctgacgc ccccgagtat aagccttggg ccctggtcat ccaggattct aacggcgaga
     4321 ataagatcaa gatgctgtct ggcggctcaa aaagaaccgc cgacggcagc gaattcgagc
     4381 ccaagaagaa gaggaaagtc taatacatct cgactgtgcc ttctagttgc cagccatctg
     4441 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     4501 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     4561 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     4621 atgcggtggg ttctatggct cgagcggccc aaaaaaagcg gttttagggg attgtaaccc
     4681 cgcagagtcc cgcaaactct ttattctagt cccttttcag ggactagaac agagacgtac
     4741 aaaaaagagc aagaagctaa aaaagattta aaaattattt ttagcgcagt taatggaaca
     4801 ggaactaaat ttaccccaaa atgtattttg tgtgctcatt tacaaccgtc tccggtgttt
     4861 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     4921 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     4981 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     5041 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     5101 tcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5161 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5221 gcgagcgcgc agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg
     5281 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5341 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5401 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5761 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5821 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5881 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5941 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6001 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6061 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6121 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6181 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6241 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6301 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6361 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6421 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6481 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6541 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6601 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6661 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6721 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6781 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6841 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6901 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6961 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7021 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7081 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7141 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7201 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7261 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7321 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7381 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7441 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7501 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7561 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7621 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7681 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7741 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7801 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7861 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7921 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7981 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccag atttaattaa
     8041 gg
//
