LOCUS       sequence-390553-        7725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome encoding N-terminal PE2max and U6 expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7725)
  AUTHORS   Davis JR, Banskota S, Levy JM, Newby GA, Wang X, Anzalone AV, Nelson
            AT, Chen PJ, Hennes AD, An M, Roh H, Randolph PB, Musunuru K, Liu DR
  TITLE     Efficient prime editing in mouse brain, liver and heart with dual 
            AAVs.
  JOURNAL   Nat Biotechnol. 2023 May 4. doi: 10.1038/s41587-023-01758-z.
  PUBMED    37142705
REFERENCE   2  (bases 1 to 7725)
  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 
            Biotechnol. 2023 May 4. doi: 10.1038/s41587-023-01758-z."
COMMENT     Sequence Label: v1em-Nterm-PE2max-U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..7725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..142)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(125..141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(134..156)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      283..738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(370..389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     580..601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        764..868
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(869..937,938..1729)
                     /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(1087..1106)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1900..2488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2389..2408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2812..2817,2818..2835,2836..2842)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2850..2866
                     /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     2855..2877
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2874..2890
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2874..2890
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2917..3046
                     /label=AAV2 ITR
     promoter        3112..3323
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3373..3393
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3394..5109
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             6814..6834
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6899..6919)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    7095..7319
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(7333..7408)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7430..7678)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7488..7507)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7658..7678)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   join(7702..7725,1..106)
                     /label=AAV2 ITR
ORIGIN
        1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
      121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
     2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagaat
     3061 tcgctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc
     3121 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct
     3181 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     3241 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     3301 acgggtttgc cgccagaaca caggaccggt gccaccatga aacggacagc cgacggaagc
     3361 gagttcgagt caccaaagaa gaagcggaaa gtcgacaaga agtacagcat cggcctggac
     3421 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     3481 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     3541 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     3601 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     3661 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     3721 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     3781 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     3841 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     3901 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     3961 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     4021 atcctgtctg ccagactgag caagagcaga aagctggaaa atctgatcgc ccagctgccc
     4081 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     4141 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     4201 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     4261 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     4321 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     4381 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     4441 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     4501 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     4561 gtgaagctga agagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     4621 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     4681 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     4741 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     4801 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     4861 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     4921 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     4981 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     5041 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     5101 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     5161 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     5221 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     5281 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     5341 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     5401 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     5461 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     5521 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     5581 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     5641 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     5701 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     5761 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     5821 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     5881 aaccggctgt ccgactacga tgtggacgct atcgtgcctc agagctttct gaaggacgac
     5941 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     6001 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     6061 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     6121 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     6181 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     6241 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     6301 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     6361 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     6421 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agtgcctgtc ctacgagaca
     6481 gagatcctga cagtggagta tggcctgctg ccaatcggca agatcgtgga gaagaggatc
     6541 gagtgtaccg tgtactctgt ggataacaat ggcaacatct atacacagcc cgtggcacag
     6601 tggcacgata ggggagagca ggaggtgttc gagtattgcc tggaggacgg cagcctgatc
     6661 agggcaacca aggaccacaa gttcatgaca gtggatggcc agatgctgcc catcgacgag
     6721 attttcgagc gggagctgga cctgatgaga gtggataacc tgcctaattc tggcggctca
     6781 aaaagaaccg ccgacggcag cgaattcgag agtcccaaga agaagaggaa agtctaagat
     6841 ctgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     6901 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     6961 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt gccacggcgg
     7021 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     7081 attccgtggt gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     7141 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     7201 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     7261 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     7321 tcgagaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     7381 tttaacttgc tatttctagc tctaaaaccg gctttttcgc gcgcgcggcg gtgtttcgtc
     7441 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat
     7501 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc
     7561 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ctaattatct
     7621 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt
     7681 cctgcccgac cttgcggccg caggaacccc tagtgatgga gttgg
//
