LOCUS       sequence-418386-        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector expressing N-terminal of SpCas9-ABE8e and tracrRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  AUTHORS   Zhang H, Kelly K, Lee J, Echeverria D, Cooper D, Panwala R, Amrani 
            N, Chen Z, Gaston N, Wagh A, Newby GA, Xie J, Liu DR, Gao G, Wolfe 
            SA, Khvorova A, Watts JK, Sontheimer EJ
  TITLE     Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery
            and genome editing in vivo.
  JOURNAL   Nucleic Acids Res. 2023 Nov 30:gkad1125. doi: 10.1093/nar/gkad1125.
  PUBMED    38033325
REFERENCE   2  (bases 1 to 7458)
  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: "Nucleic 
            Acids Res. 2023 Nov 30:gkad1125. doi: 10.1093/nar/gkad1125."
COMMENT     Sequence Label: pEJS-HZ96 AAV-SpABE8e-N-terminus_tracrRNA
FEATURES             Location/Qualifiers
     source          1..7458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        33..281
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     33..53
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     204..223
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    360..584
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     760..780
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(845..865)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1214..2929)
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             complement(3524..3544)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          3598..3825
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(3826..4103)
                     /label=chicken beta-actin promoter
     enhancer        4105..4390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     repeat_region   4413..4542
                     /label=AAV2 ITR
     primer_bind     complement(4569..4585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4569..4585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4582..4604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4593..4609
                     /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(4617..4623,4624..4641,4642..4647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4662..4683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4971..5559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5051..5070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5730..6521,6522..6590))
                     /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     6353..6372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6591..6695)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6721..7176)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6858..6879)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7070..7089
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7303..7325
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7317..7334
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7318..7334
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   join(7353..7458,1..24)
                     /label=AAV2 ITR
ORIGIN
        1 ccaactccat cactaggggt tcctgcggcc gcgagggcct atttcccatg attccttcat
       61 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
      121 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      181 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      241 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgagcatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctcgagc
      361 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct
      421 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat
      481 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac
      541 gggggagggg caaacaacag atggctggca actagaaggc acagtcgcac cacggaattg
      601 tcagtgccca acagccgagc ccctgtccag cagcgggcaa ggcaggcggc gatgagttcc
      661 gccgtggcaa gaactaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa
      721 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca
      781 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attatcagat
      841 cttacacctt tctctttttc ttgggctcaa actcactccc gtctgctgtt cttttactgc
      901 ctccgctatt aggcaggtta tccactctca tcaggtccag ctcccgctcg aaaatctcgt
      961 cgatgggcag catctggcca tccactgtca tgaacttgtg gtccttggtt gccctgatca
     1021 ggctgccgtc ctccaggcaa tactcgaaca cctcctgctc tcccctatcg tgccactgtg
     1081 ccacgggctg tgtatagatg ttgccattgt tatccacaga gtacacggta cactcgatcc
     1141 tcttctccac gatcttgccg attggcagca ggccatactc cactgtcagg atctctgtct
     1201 cgtaggacag gcactcgatt ttcttgaagt agtcctcttt cagctgcttc acggtcactt
     1261 tccggttggt cttgaacagc aggtccacga tggccttttt ctgctcgccg ctcaggaagg
     1321 cgggctttct cattccctcg gtcacgtatt tcactttggt cagctcgtta tacacggtga
     1381 agtactcgta cagcaggctg tgcttgggca gcaccttctc gttgggcagg ttcttatcga
     1441 agttggtcat ccgctcgatg aagctctggg cggaagcgcc cttgtccacc acttcctcga
     1501 agttccaggg ggtgatggtt tcctcgctct ttctggtcat ccaggcgaat ctgctgtttc
     1561 ccctggccag agggcccacg tagtagggga tgcggaaggt caggatcttc tcgatctttt
     1621 cccggttgtc cttcaggaat gggtaaaaat cttcctgccg ccgcagaatg gcgtgcagct
     1681 ctcccaggtg gatctggtgg gggatgctgc cgttgtcgaa ggtccgctgc ttccgcagca
     1741 ggtcctctct gttcagcttc acgagcagtt cctcggtgcc gtccatcttt tccaggatgg
     1801 gcttgatgaa cttgtagaac tcttcctggc tggctccgcc gtcaatgtag ccggcgtagc
     1861 cgttcttgct ctggtcgaag aaaatctctt tgtacttctc aggcagctgc tgccgcacga
     1921 gagctttcag cagggtcagg tcctggtggt gctcgtcgta tctcttgatc atagaggcgc
     1981 tcaggggggc cttggtgatc tcggtgttca ctctcaggat gtcgctcagc aggatggcgt
     2041 cggacaggtt cttggcggcc agaaacaggt cggcgtactg gtcgccgatc tgggccagca
     2101 ggttgtccag gtcgtcgtcg taggtgtcct tgctcagctg cagtttggca tcctcggcca
     2161 ggtcgaagtt gctcttgaag ttgggggtca ggcccaggct cagggcaatc aggtttccga
     2221 acaggccatt cttcttctcg ccgggcagct gggcgatcag attttccagc cgtctgctct
     2281 tgctcagtct ggcagacagg atggccttgg cgtccacgcc gctggcgttg atggggtttt
     2341 cctcgaacag ctggttgtag gtctgcacca gctggatgaa cagcttgtcc acgtcgctgt
     2401 tgtcggggtt caggtcgccc tcgatcagga agtggccccg gaacttgatc atgtgggcca
     2461 gggccagata gatcagccgc aggtcggcct tgtcggtgct gtccaccagt ttctttctca
     2521 ggtggtagat ggtggggtac ttctcgtggt aggccacctc gtccacgatg ttgccgaaga
     2581 tggggtgccg ctcgtgcttc ttatcctctt ccaccaggaa ggactcttcc agtctgtgga
     2641 agaagctgtc gtccaccttg gccatctcgt tgctgaagat ctcttgcaga tagcagatcc
     2701 ggttcttccg tctggtgtat cttcttctgg cggttctctt cagccgggtg gcctcggctg
     2761 tttcgccgct gtcgaacagc agggctccga tcaggttctt cttgatgctg tgccggtcgg
     2821 tgttgcccag caccttgaat ttcttgctgg gcaccttgta ctcgtcggtg atcacggccc
     2881 agcccacaga gttggtgccg atggccaggc cgatgctgta cttcttgtct gaccccccgc
     2941 tgctgccccc gctgctttca ggtgttgcgc tctcgcttgt gccaggtgtc tcagagccag
     3001 aggagcctcc gctagatcct ccggagttga tggagctctg ggccttcttc tgagcattga
     3061 acacctgtct aggcatccga tagaaatcgc acagcagggc ggcacattca tctgccagga
     3121 ttccctcggt aatttcgacg cggtgattca tgccggggta gttcagcacg ttcatcaggg
     3181 agcctgcggc gcctcttttt gagttcctca cgccaaacac cacgcggccg atcctagagt
     3241 ggatcatggc gccggcgcac atcacgcaag gctcgaatgt cacgtacagg gtggcgtcaa
     3301 tcagtctgta gttctgcatg accaggccgc cctgtctcag ggccataatt tcggcatggg
     3361 ctgttgggtc gtgcaggccg atggctctgt tccagccctc gccgatcact ctattgttca
     3421 gcaccagcac ggctcccaca ggcacctccc tctcatcccg tgccctcttg gccagggtca
     3481 gggcatgtct catccagtac tcgtgggaaa actccacctc agagactttc cgcttcttct
     3541 ttggtgactc gaactcgctt ccatcggctg tccgtttcat ggtggcaccg gtccaacctg
     3601 aaaaaaagtg atttcaggca ggtgctccag gtaattaaac attaataccc caccaaccaa
     3661 ccatccctta aacccttacc tcttgctcag ctaattacag cccggaggag aagggccgtc
     3721 ccgcccgctc acctgtggga gtaacgcggt cagtcagagc cggggcgggc ggcgcgaggc
     3781 ggcggcggag cggggcacgg ggcgaaggca gcgcgcagcg actcccgccc gccgcgcgct
     3841 tcgcttttta tagggccgcc gccgccgccg cctcgccata aaaggaaact ttcggagcgc
     3901 gccgctctga ttggctgccg ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg
     3961 ccccgccccg cctggcgcgc gccccccccc cccccccgcc cccatcgctg cacaaaataa
     4021 ttaaaaaata aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa
     4081 gcagaacgtg gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg
     4141 ccaagtagga aagtcccata aggtcatgta ctgggcacaa tgccaggcgg gccatttacc
     4201 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     4261 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     4321 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     4381 ttatgtaacg ggtaccctga tctagaggcc gcaggaaccc ctagtgatgg agttggccac
     4441 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4501 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agccttaatt aaatctggcg
     4561 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4621 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4681 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4741 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4801 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4861 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4921 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4981 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5041 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5101 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5161 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5221 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5281 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5341 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5401 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5461 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5521 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5581 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5641 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5701 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5761 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5821 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5881 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5941 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6001 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6061 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6121 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6181 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6241 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6301 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6361 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6421 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6481 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6541 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6601 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6661 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6721 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6781 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     6841 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6901 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     6961 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     7021 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7081 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7141 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     7201 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     7261 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     7321 aaacgacggc cagtgaatta ggttaattaa ggctgcgcgc tcgctcgctc actgaggccg
     7381 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     7441 cgcgcagaga gggagtgg
//
