LOCUS       sequence-390551-        7690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome encoding C-terminal PE2max and U6 expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  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 7690)
  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-Cterm-PE2max-U6-pegRNA
FEATURES             Location/Qualifiers
     source          1..7690
                     /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             4588..4608
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6709..6729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6794..6814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    6990..7214
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(7298..7373)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7395..7643)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7453..7472)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7623..7643)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   join(7667..7690,1..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 gtcatcaaga ttgctacacg gaaatacctg
     3421 ggaaagcaga acgtgtacga catcggcgtg gagcgggatc acaacttcgc cctgaagaat
     3481 ggctttatcg ccagcaattg tttcaacgaa atcggcaagg ctaccgccaa gtacttcttc
     3541 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     3601 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     3661 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     3721 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     3781 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     3841 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     3901 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     3961 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4021 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4081 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4141 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4201 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4261 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     4321 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4381 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4441 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     4501 cggatcgacc tgtctcagct gggaggtgac tccggcggaa gctctggtgg cagcaagcgg
     4561 accgccgacg gctctgaatt cgagagccct aagaagaaaa gaaaggtgag cggaggctct
     4621 agcggcggaa gcaccctgaa cattgaagac gagtatagac tgcatgaaac aagcaaggaa
     4681 cccgacgtgt ccctgggctc cacctggctg tccgactttc cccaggcctg ggccgagaca
     4741 ggaggaatgg gcctggccgt gcggcaggca cccctgatca tccctctgaa ggccacctct
     4801 acacccgtga gcatcaagca gtaccctatg tctcaggagg ccagactggg catcaagcct
     4861 cacatccaga ggctgctgga ccagggcatc ctggtgccat gccagagccc ctggaacaca
     4921 ccactgctgc ccgtgaagaa gccaggcacc aatgactata gacccgtgca ggatctgaga
     4981 gaggtgaaca agagggtgga ggatatccac cccaccgtgc ccaaccctta caatctgctg
     5041 tccggcctgc ccccttctca ccagtggtat acagtgctgg acctgaagga tgccttcttt
     5101 tgtctgagac tgcaccctac cagccagcca ctgttcgcct ttgagtggag ggaccctgag
     5161 atgggcatct ctggccagct gacctggaca cgcctgcctc agggcttcaa gaatagccca
     5221 acactgttta acgaggccct gcaccgcgac ctggcagatt tccggatcca gcacccagat
     5281 ctgatcctgc tgcagtacgt ggacgatctg ctgctggccg ccaccagcga gctggattgc
     5341 cagcagggaa cacgcgccct gctgcagacc ctgggaaacc tgggatatag ggcatccgcc
     5401 aagaaggccc agatctgtca gaagcaggtg aagtacctgg gctatctgct gaaggagggc
     5461 cagagatggc tgacagaggc caggaaggag acagtgatgg gccagccaac acccaagacc
     5521 ccaagacagc tgagggagtt cctgggcaaa gcaggatttt gcaggctgtt catcccagga
     5581 ttcgcagaga tggcagcacc tctgtaccca ctgaccaagc cgggcaccct gtttaattgg
     5641 ggccctgacc agcagaaggc ctatcaggag atcaagcagg ccctgctgac agcaccagcc
     5701 ctgggcctgc cagacctgac caagcctttc gagctgtttg tggatgagaa gcagggctac
     5761 gccaagggcg tgctgaccca gaagctggga ccatggagac ggcccgtggc ctatctgtcc
     5821 aagaagctgg acccagtggc agcaggatgg ccaccatgcc tgaggatggt ggcagcaatc
     5881 gccgtgctga caaaggatgc cggcaagctg accatgggac agccactggt catcctggca
     5941 ccacacgcag tggaggccct ggtgaagcag cctccagatc gctggctgtc taacgcccgg
     6001 atgacacact accaggccct gctgctggac accgatcgcg tgcagtttgg ccctgtggtg
     6061 gccctgaatc cagccaccct gctgcctctg ccagaggagg gcctgcagca caactgtctg
     6121 gacatcctgg cagaggcaca cggaacaagg ccagacctga ccgatcagcc cctgcctgac
     6181 gccgatcaca catggtatac cgatggaagc tccctgctgc aggagggcca gaggaaggca
     6241 ggagcagcag tgaccacaga gacagaagtg atctgggcca aggccctgcc agcaggcaca
     6301 tccgcccagc gggccgagct gatcgccctg acccaggccc tgaagatggc cgagggcaag
     6361 aagctgaacg tgtacacaga ctccagatat gccttcgcca ccgcacacat ccacggagag
     6421 atctacaggc gccggggctg gctgacctct gagggcaagg agatcaagaa caaggatgag
     6481 atcctggccc tgctgaaggc cctgtttctg cccaagcggc tgagcatcat ccactgtcct
     6541 ggacaccaga agggacactc cgccgaggca aggggcaatc ggatggccga ccaggccgcc
     6601 agaaaggctg ctattactga aactcccgac acttccactc tgctgattga aaactcctcc
     6661 ccttctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagtctcc caagaagaag
     6721 aggaaagtct aagatctgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     6781 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     6841 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag
     6901 ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6961 tgttgggcac tgacaattcc gtggtgcgac tgtgccttct agttgccagc catctgttgt
     7021 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     7081 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     7141 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     7201 ggtgggctct atggctcgag aaaaaaattc tagttggttt aacgcgtaac tagatagaac
     7261 cgcggtgtgt ttctggagct gttcgccctg gcatcttgca ccgactcggt gccacttttt
     7321 caagttgata acggactagc cttattttaa cttgctattt ctagctctaa aacgcgcgaa
     7381 cagctccagc ccgcggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     7441 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     7501 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     7561 acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     7621 ggaatcatgg gaaataggcc ctcttcctgc ccgaccttgc ggccgcagga acccctagtg
     7681 atggagttgg
//
