LOCUS       sequence-390541-        7777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome encoding C-terminal PE2max and U6 expression cassettes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7777)
  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 7777)
  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: v3em-Cterm-PE2max-∆RNaseH-dualU6
FEATURES             Location/Qualifiers
     source          1..7777
                     /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
     enhancer        3069..3354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3356..3632
                     /label=chicken beta-actin promoter
     intron          3633..3860
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3914..3934
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5129..5149
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6710..6730
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     6750..6769
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_RNA        complement(6888..6963)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(6986..7299)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(7047..7067)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        complement(7385..7460)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7482..7730)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7540..7559)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7710..7730)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   join(7754..7777,1..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 cctctagatc
     3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
     3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg ggggggcgcg
     3481 cgccaggsgg gggggggsgg ggggaggggg gggggggggs gaggsggaga ggtgsggcgg
     3541 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     3601 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
     3661 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
     3721 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
     3781 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
     3841 ctgaaatcac tttttttcag gttggaccgg tgccaccatg aaacggacag ccgacggaag
     3901 cgagttcgag tcaccaaaga agaagcggaa agtcatcaag attgctacac ggaaatacct
     3961 gggaaagcag aacgtgtacg acatcggcgt ggagcgggat cacaacttcg ccctgaagaa
     4021 tggctttatc gccagcaatt gtttcaacga aatcggcaag gctaccgcca agtacttctt
     4081 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     4141 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     4201 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     4261 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     4321 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     4381 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     4441 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     4501 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     4561 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     4621 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     4681 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     4741 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     4801 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     4861 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     4921 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     4981 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     5041 acggatcgac ctgtctcagc tgggaggtga ctccggcgga agctctggtg gcagcaagcg
     5101 gaccgccgac ggctctgaat tcgagagccc taagaagaaa agaaaggtga gcggaggctc
     5161 tagcggcgga agcaccctga acattgaaga cgagtataga ctgcatgaaa caagcaagga
     5221 acccgacgtg tccctgggct ccacctggct gtccgacttt ccccaggcct gggccgagac
     5281 aggaggaatg ggcctggccg tgcggcaggc acccctgatc atccctctga aggccacctc
     5341 tacacccgtg agcatcaagc agtaccctat gtctcaggag gccagactgg gcatcaagcc
     5401 tcacatccag aggctgctgg accagggcat cctggtgcca tgccagagcc cctggaacac
     5461 accactgctg cccgtgaaga agccaggcac caatgactat agacccgtgc aggatctgag
     5521 agaggtgaac aagagggtgg aggatatcca ccccaccgtg cccaaccctt acaatctgct
     5581 gtccggcctg cccccttctc accagtggta tacagtgctg gacctgaagg atgccttctt
     5641 ttgtctgaga ctgcacccta ccagccagcc actgttcgcc tttgagtgga gggaccctga
     5701 gatgggcatc tctggccagc tgacctggac acgcctgcct cagggcttca agaatagccc
     5761 aacactgttt aacgaggccc tgcaccgcga cctggcagat ttccggatcc agcacccaga
     5821 tctgatcctg ctgcagtacg tggacgatct gctgctggcc gccaccagcg agctggattg
     5881 ccagcaggga acacgcgccc tgctgcagac cctgggaaac ctgggatata gggcatccgc
     5941 caagaaggcc cagatctgtc agaagcaggt gaagtacctg ggctatctgc tgaaggaggg
     6001 ccagagatgg ctgacagagg ccaggaagga gacagtgatg ggccagccaa cacccaagac
     6061 cccaagacag ctgagggagt tcctgggcaa agcaggattt tgcaggctgt tcatcccagg
     6121 attcgcagag atggcagcac ctctgtaccc actgaccaag ccgggcaccc tgtttaattg
     6181 gggccctgac cagcagaagg cctatcagga gatcaagcag gccctgctga cagcaccagc
     6241 cctgggcctg ccagacctga ccaagccttt cgagctgttt gtggatgaga agcagggcta
     6301 cgccaagggc gtgctgaccc agaagctggg accatggaga cggcccgtgg cctatctgtc
     6361 caagaagctg gacccagtgg cagcaggatg gccaccatgc ctgaggatgg tggcagcaat
     6421 cgccgtgctg acaaaggatg ccggcaagct gaccatggga cagccactgg tcatcctggc
     6481 accacacgca gtggaggccc tggtgaagca gcctccagat cgctggctgt ctaacgcccg
     6541 gatgacacac taccaggccc tgctgctgga caccgatcgc gtgcagtttg gccctgtggt
     6601 ggccctgaat ccagccaccc tgctgcctct gccagaggag ggcctgcagc acaactgtct
     6661 ggactctggc ggctcaaaaa gaaccgccga cggcagcgaa ttcgagtctc ccaagaagaa
     6721 gaggaaagtc taagtcgacc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     6781 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     6841 ttcaggggga gatgtgggag gttttttaaa gcaagcttgc aaaaaaagca ccgactcggt
     6901 gccacttttt caagttgata acggactagc cttattttaa cttgctattt ctagctctaa
     6961 aaccggcttt ttcgcgcgcg cggcaaacaa ggcttttctc caagggatat ttatagtctc
     7021 aaaacacaca attactttac agttagggtg agtttccttt tgtgctgttt tttaaaataa
     7081 taatttagta tttgtatctc ttatagaaat ccaagcctat catgtaaaat gtagctagta
     7141 ttaaaaagaa cagattatct gtcttttatc gcacattaag cctctatagt tactaggaaa
     7201 tattatatgc aaattaaccg gggcagggga gtagccgagc ttctcccaca agtctgtgcg
     7261 agggggccgg cgcgggccta gagatggcgg cgtcggatcg cgaattcaaa aaaattctag
     7321 ttggtttaac gcgtaactag atagaaccgc ggtgtgtttc tggagctgtt cgggctggca
     7381 tcttgcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
     7441 gctatttcta gctctaaaac gcgcgaacag ctccagcccg cggtgtttcg tcctttccac
     7501 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
     7561 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
     7621 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca
     7681 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccg
     7741 accttgcggc cgcaggaacc cctagtgatg gagttgg
//
