LOCUS       sequence-418387-        7332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector expressing C-terminus of SpCas9-ABE8e with tracrRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7332)
  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 7332)
  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-HZ100 AAV-SpABE8e-C_terminus-tracrRNA
FEATURES             Location/Qualifiers
     source          1..7332
                     /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(908..3292)
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(3398..3418)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          3472..3699
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(3700..3977)
                     /label=chicken beta-actin promoter
     enhancer        3979..4264
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     repeat_region   4287..4416
                     /label=AAV2 ITR
     primer_bind     complement(4443..4459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4443..4459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4456..4478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4467..4483
                     /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(4491..4497,4498..4515,4516..4521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4536..4557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4845..5433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4925..4944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5604..6395,6396..6464))
                     /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     6227..6246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6465..6569)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6595..7050)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6732..6753)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6944..6963
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7177..7199
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7191..7208
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7192..7208
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   join(7227..7332,1..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 cttagacttt cctcttcttc ttgggctcga attcgctgcc gtcggcggtt ctttttgagc
      901 cgccagagtc acctcccagc tgagacaggt cgatccgtgt ctcgtacagg ccggtgatgc
      961 tctggtggat cagggtggcg tccagcacct ctttggtgct ggtgtacctc ttccggtcga
     1021 tggtggtgtc aaagtacttg aaggcggcag gggctcccag attggtcagg gtaaacaggt
     1081 ggatgatatt ctcggcctgc tctctgatgg gcttatcccg gtgcttgttg taggcggaca
     1141 gcactttgtc cagattagcg tcggccagga tcactctctt ggagaactcg ctgatctgct
     1201 cgatgatctc gtccaggtag tgcttgtgct gttccacaaa cagctgtttc tgctcattat
     1261 cctcggggga gcccttcagc ttctcatagt ggctggccag gtacaggaag ttcacatatt
     1321 tggagggcag ggccagttcg tttcccttct gcagttcgcc ggcagaggcc agcattctct
     1381 tccggccgtt ttccagctcg aacagggagt acttaggcag cttgatgatc aggtcctttt
     1441 tcacttcttt gtagcccttg gcttccagaa agtcgatggg attcttctcg aagctgcttc
     1501 tttccatgat ggtgatcccc agcagctctt tcacactctt cagtttcttg gacttgccct
     1561 tttccacttt ggccaccacc agcacagaat aggccacggt ggggctgtcg aagccgccgt
     1621 acttcttagg gtcccagtcc ttctttctgg cgatcagctt atcgctgttc ctcttgggca
     1681 ggatagactc tttgctgaag ccgcctgtct gcacctcggt ctttttcacg atattcactt
     1741 ggggcatgct cagcactttc cgcacggtgg caaaatcccg gcccttatcc cacacgatct
     1801 ccccggtttc gccgtttgtc tcgatcagag gccgcttccg gatctcgccg ttggccaggg
     1861 taatctcggt cttgaaaaag ttcatgatgt tgctgtagaa gaagtacttg gcggtagcct
     1921 tgccgatttc ctgctcgctc ttggcgatca tcttccgcac gtcgtacacc ttgtagtcgc
     1981 cgtacacgaa ctcgctttcc agcttagggt actttttgat cagggcggtt cccacgacgg
     2041 cgttcaggta ggcgtcgtgg gcgtggtggt agttgttgat ctcgcgcact ttgtaaaact
     2101 ggaaatcctt ccggaaatcg gacaccagct tggacttcag ggtgatcact ttcacttccc
     2161 ggatcagctt gtcattctcg tcgtacttag tgttcatccg ggagtccagg atctgtgcca
     2221 cgtgctttgt gatctgccgg gtttccacca gctgtctctt gatgaagccg gccttatcca
     2281 gttcgctcag gccgcctctc tcggccttgg tcagattgtc gaactttctc tgggtaatca
     2341 gcttggcgtt cagcagctgc cgccagtagt tcttcatctt cttcacgacc tcttcggagg
     2401 gcacgttgtc gctcttgccc cggttcttgt cgcttctggt cagcaccttg ttgtcgatgg
     2461 agtcgtcctt cagaaagctc tgaggcacga tatggtccac atcgtagtcg gacagccggt
     2521 tgatgtccag ttcctggtcc acgtacatat cccgcccatt ctgcaggtag tacaggtaca
     2581 gcttctcgtt ctgcagctgg gtgttttcca cggggtgttc tttcaggatc tggctgccca
     2641 gctctttgat gccctcttcg atccgcttca ttctctcgcg gctgttcttc tgtcccttct
     2701 gggtggtctg gttctctctg gccatttcga tcacgatgtt ctcgggcttg tgccggccca
     2761 tcactttcac gagctcgtcc accaccttca ctgtctgcag gatgcccttc ttaatggcgg
     2821 ggctgccggc cagattggca atgtgctcgt gcaggctatc gccctggccg gacacctggg
     2881 ctttctggat gtcctcttta aaggtcaggc tgtcgtcgtg gatcagctgc atgaagtttc
     2941 tgttggcgaa gccgtcggac ttcaggaaat ccaggattgt cttgccggac tgcttgtccc
     3001 ggatgccgtt gatcagcttc cggctcagcc tgccccagcc ggtgtatctc cgccgcttca
     3061 gctgcttcat cactttgtcg tcgaacaggt gggcataggt tttcagccgt tcctcgatca
     3121 tctctctgtc ctcaaacagt gtcagggtca gcacgatatc ttccagaatg tcctcgtttt
     3181 cctcattgtc caggaagtcc ttgtccttga taattttcag cagatcgtgg tatgtgccca
     3241 gggaggcgtt gaaccgatct tccacgccgg agatttccac ggagtcgaag caattgctgg
     3301 cgataaagcc attcttcagg gcgaagttgt gatcccgctc cacgccgatg tcgtacacgt
     3361 tctgctttcc caggtatttc cgtgtagcaa tcttgatgac tttccgcttc ttctttggtg
     3421 actcgaactc gcttccatcg gctgtccgtt tcatggtggc accggtccaa cctgaaaaaa
     3481 agtgatttca ggcaggtgct ccaggtaatt aaacattaat accccaccaa ccaaccatcc
     3541 cttaaaccct tacctcttgc tcagctaatt acagcccgga ggagaagggc cgtcccgccc
     3601 gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg aggcggcggc
     3661 ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg cgcttcgctt
     3721 tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga gcgcgccgct
     3781 ctgattggct gccgccgcac ctctccgcct cgccccgccc cgcccctcgc cccgccccgc
     3841 cccgcctggc gcgcgccccc cccccccccc cgcccccatc gctgcacaaa ataattaaaa
     3901 aataaataaa tacaaaattg ggggtgggga ggggggggag atggggagag tgaagcagaa
     3961 cgtggggctc acctcgacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     4021 aggaaagtcc cataaggtca tgtactgggc acaatgccag gcgggccatt taccgtcatt
     4081 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     4141 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactattga
     4201 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     4261 aacgggtacc ctgatctaga ggccgcagga acccctagtg atggagttgg ccactccctc
     4321 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4381 tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaatct ggcgtaatca
     4441 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4501 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4561 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4621 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4681 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4741 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4801 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4861 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4921 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4981 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5041 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5101 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5161 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5221 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5281 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5341 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5401 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5461 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5521 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5581 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5641 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5701 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5761 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5821 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5881 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5941 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6001 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6061 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6121 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6181 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6241 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6301 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6361 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6421 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6481 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6541 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6601 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6661 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6721 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6781 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6841 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6901 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6961 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7021 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     7081 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     7141 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     7201 cggccagtga attaggttaa ttaaggctgc gcgctcgctc gctcactgag gccgcccggg
     7261 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     7321 gagagggagt gg
//
