LOCUS       sequence-370608-        7689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV1 for expression of nickase SpCas9(H840A) for Split-PE use.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   Grunewald J, Miller BR, Szalay RN, Cabeceiras PK, Woodilla CJ, Holtz
            EJB, Petri K, Joung JK
  TITLE     Engineered CRISPR prime editors with compact, untethered reverse 
            transcriptases.
  JOURNAL   Nat Biotechnol. 2023 Mar;41(3):337-343. doi: 
            10.1038/s41587-022-01473-1. Epub 2022 Sep 26.
  PUBMED    36163548
REFERENCE   2  (bases 1 to 7689)
  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"; doi: 
            "10.1038/s41587-022-01473-1"; journalName: "Nat Biotechnol"; date: 
            "2023-03"; volume: "41"; issue: "3"; pages: "337-343"
COMMENT     Sequence Label: AAV1 for Split-PE, SpCas9 nickase (PKC1001)
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   24..153
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        198..409
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             422..690
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             755..775
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(776..796,797..799,800..802,803..3286,3287..3292,
                     3293..3358,3359..3361,3362..4876)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4880..4900
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4919..4967
                     /note="synthetic polyadenylation signal"
     repeat_region   4975..5115
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4975..5104
                     /label=AAV2 ITR
     rep_origin      5190..5645
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5277..5296)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5487..5508
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5662..5681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5781..5803
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5841..5859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5927..6031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6032..6100,6101..6892)
                     /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(6250..6269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7063..7651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7552..7571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
       61 cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
      121 agggagtggc caactccatc actaggggtt cctgcggcct ctagaaagga tctgcgatcg
      181 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      241 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      301 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      361 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
      421 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
      481 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
      541 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
      601 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
      661 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta cagccaccat
      721 gaaacggaca gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcgacaa
      781 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
      841 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
      901 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
      961 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     1021 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     1081 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatctttg gcaatatcgt
     1141 ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt
     1201 agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa
     1261 atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa
     1321 actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc
     1381 atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga
     1441 aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc
     1501 cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa
     1561 gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg
     1621 cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag
     1681 tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa
     1741 gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact
     1801 gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat
     1861 tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat
     1921 ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg
     1981 cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct
     2041 caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat
     2101 cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc
     2161 gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga
     2221 taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc
     2281 taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga
     2341 gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga
     2401 gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca
     2461 gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt
     2521 ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga
     2581 caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct
     2641 tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt
     2701 cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc
     2761 aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct
     2821 taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg actctctcac
     2881 ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca
     2941 catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt
     3001 cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat
     3061 ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag
     3121 gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa
     3181 cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta
     3241 cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggctg ctatcgtgcc
     3301 ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaagc
     3361 tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg
     3421 gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc
     3481 tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga
     3541 gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta
     3601 cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt
     3661 ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca
     3721 tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa
     3781 gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc
     3841 aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat
     3901 gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat
     3961 cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt
     4021 ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg
     4081 aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa
     4141 aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt
     4201 actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact
     4261 gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga
     4321 ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct
     4381 ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg
     4441 taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga
     4501 aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa
     4561 acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc
     4621 cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag
     4681 ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc
     4741 cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct
     4801 ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc
     4861 tcagctcggt ggagacagcc ccaagaagaa gagaaaggtg gaggccagct aagaattcaa
     4921 taaaagatct ttattttcat tagatctgtg tgttggtttt ttgtgtgcgg ccgcaggaac
     4981 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5041 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5101 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5161 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     5221 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     5281 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5341 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5401 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5461 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5521 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     5581 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     5641 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5701 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5761 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5821 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5881 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5941 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6001 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6061 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6721 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     6781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     7261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     7381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7681 cctggcctt
//
