LOCUS       sequence-483897-        7658 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  AAV genome: expresses the C-terminal of v5 AAV-CBE from the Cbh 
            promoter, and U6-sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7658)
  AUTHORS   Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan 
            LW, Comander J, Liu Q, Liu DR
  TITLE     Cytosine and adenine base editing of the brain, liver, retina, heart
            and skeletal muscle of mice via adeno-associated viruses.
  JOURNAL   Nat Biomed Eng. 2020 Jan;4(1):97-110. doi: 
            10.1038/s41551-019-0501-5. Epub 2020 Jan 14.
  PUBMED    31937940
REFERENCE   2  (bases 1 to 7658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41551-019-0501-5"; journalName: "Nat Biomed Eng"; date: 
            "2020-01"; volume: "4"; issue: "1"; pages: "97-110"
COMMENT     Sequence Label: Cbh_v5 AAV-CBE C-terminal
FEATURES             Location/Qualifiers
     source          1..7658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     enhancer        153..438
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        440..717
                     /label=chicken beta-actin promoter
     intron          718..945
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             999..1019
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1125..3509
                     /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             3540..3788
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3831..3851
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3916..3936)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4112..4336
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4350..4425)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4447..4695)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4505..4524)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4675..4695)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4719..4848
                     /label=AAV2 ITR
     primer_bind     complement(4867..4884)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4867..4883)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4876..4898)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5025..5480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5112..5131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5322..5343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5506..5610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5611..5679,5680..6471)
                     /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(5829..5848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6642..7230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7131..7150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7518..7539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7554..7559,7560..7577,7578..7584)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7592..7608
                     /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     7597..7619
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7616..7632
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7616..7632
                     /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 gcggcctcta gatcagggta cccgttacat aacttacggt aaatggcccg
      181 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
      241 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      301 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      361 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      421 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      481 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      541 ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      601 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      661 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      721 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      781 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      841 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
      901 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca
      961 ccatgaaacg gacagccgac ggaagcgagt tcgagtcacc aaagaagaag cggaaagtca
     1021 tcaagattgc tacacggaaa tacctgggaa agcagaacgt gtacgacatc ggcgtggagc
     1081 gggatcacaa cttcgccctg aagaatggct ttatcgccag caattgcttc gactccgtgg
     1141 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     1201 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     1261 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     1321 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     1381 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     1441 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     1501 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     1561 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     1621 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     1681 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     1741 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     1801 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     1861 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     1921 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     1981 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     2041 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     2101 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     2161 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     2221 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     2281 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     2341 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     2401 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     2461 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     2521 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     2581 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     2641 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     2701 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     2761 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     2821 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     2881 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     2941 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     3001 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     3061 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     3121 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     3181 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     3241 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     3301 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     3361 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     3421 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     3481 ggatcgacct gtctcagctg ggaggtgaca gcggcgggag cggcgggagc ggggggagca
     3541 ctaatctgag cgacatcatt gagaaggaga ctgggaaaca gctggtcatt caggagtcca
     3601 tcctgatgct gcctgaggag gtggaggaag tgatcggcaa caagccagag tctgacatcc
     3661 tggtgcacac cgcctacgac gagtccacag atgagaatgt gatgctgctg acctctgacg
     3721 cccccgagta taagccttgg gccctggtca tccaggattc taacggcgag aataagatca
     3781 agatgctgag cggaggatcc aaaagaaccg ccgacggcag cgaattcgag cccaagaaga
     3841 agaggaaagt ctaagatctg ataatcaacc tctggattac aaaatttgtg aaagattgac
     3901 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3961 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4021 agttcttgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4081 gctgttgggc actgacaatt ccgtggtgcg actgtgcctt ctagttgcca gccatctgtt
     4141 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4201 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4261 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4321 gcggtgggct ctatggctcg agaaaaaaag caccgactcg gtgccacttt ttcaagttga
     4381 taacggacta gccttatttt aacttgctat ttctagctct aaaaccgaga cgcattaatc
     4441 gtctcgggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca
     4501 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc
     4561 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa
     4621 attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat
     4681 gggaaatagg ccctcttcct gcccgacctt gcggccgcag gaacccctag tgatggagtt
     4741 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4801 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc
     4861 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4921 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4981 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5041 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5101 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5161 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5221 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5281 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5341 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5401 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5461 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5521 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5581 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5641 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5701 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5761 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5821 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5881 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5941 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6001 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6061 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6121 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6181 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6241 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6301 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6361 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6421 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6481 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6541 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6601 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6661 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6721 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6781 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6841 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6901 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6961 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7021 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7081 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7141 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7201 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7261 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     7321 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     7381 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     7441 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7501 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     7561 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     7621 aacagctatg accatgatta cgccagattt aattaagg
//
