LOCUS       sequence-372717-        6887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV2 for expression of pegRNA and ngRNA (HEKs3, CTT insertion) + 
            MMLV-RT(dRH)-P2A-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6887)
  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 6887)
  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: AAV2 for Split-PE, gRNAs + MMLV-RT(dRH)-P2A-eG...
FEATURES             Location/Qualifiers
     source          1..6887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        251..499
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     251..271
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     422..441
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        520..595
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        636..850
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     771..790
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        879..954
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1017..1228
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1318..1338
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2869..2889
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2899..2955
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(2956..2958,2959..2961,2962..3675)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2956..2958,2959..2961,2962..3672)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3001..3022)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3262..3281)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3609..3630
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3768..3788)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4148..4288
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4148..4277
                     /label=AAV2 ITR
     rep_origin      4363..4818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4450..4469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4660..4681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4835..4854)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4954..4976
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5014..5032)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5100..5204
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5205..5273,5274..6065)
                     /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(5423..5442)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6236..6824
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6725..6744
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6886..6887,1..128)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
       61 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
      121 gggttcctgc ggccgcagat gctgcacact tgtggactac tagtaagctt ggatcttgaa
      181 gaagctgcag gaggtgctgg aggggaagtg gtccggatcc gatcagtgtg agggagtgta
      241 aagctggttt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
      301 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
      361 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
      421 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
      481 gtggaaagga cgaaacaccg gcccagactg agcacgtgag ttttagagct agaaatagca
      541 agttaaaata aggctagtcc gttatcaact tgaaaaagtg ggaccgagtc ggtcctctgc
      601 catcaaagcg tgctcagtct gtttttttgg aattcgaacg ctgacgtcat caacccgctc
      661 caaggaatcg cgggcccagt gtcactaggc gggaacaccc agcgcgcgtg cgccctggca
      721 ggaagatggc tgtgagggac aggggagtgg cgccctgcaa tatttgcatg tcgctatgtg
      781 ttctgggaaa tcaccataaa cgtgaaatgt ctttggattt gggaatctta taagttctgt
      841 atgagaccac tttttcccgt caaccagtat cccggtgcgt tttagagcta gaaatagcaa
      901 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      961 tctaactcga acgctagctg tgcgatcgtt ttcgagtggc tccggtgccc gtcagtgggc
     1021 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     1081 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     1141 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     1201 tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggacccgac attagcgcta
     1261 cagcttaagc gggccgccac catgaaacgg acagccgacg gaagcgagtt cgagtcacca
     1321 aagaagaagc ggaaagtcac cctaaatata gaagatgagt atcggctaca tgagacctca
     1381 aaagagccag atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg
     1441 gaaaccgggg gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca
     1501 acctctaccc ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc
     1561 aagccccaca tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg
     1621 aacacgcccc tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat
     1681 ctgagagaag tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac
     1741 ctcttgagcg ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc
     1801 tttttctgcc tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat
     1861 ccagagatgg gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac
     1921 agtcccaccc tgtttaatga ggcactgcac agagacctag cagacttccg gatccagcac
     1981 ccagacttga tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta
     2041 gactgccaac aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc
     2101 tcggccaaga aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa
     2161 gagggtcaga gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg
     2221 aagacccctc gacaactaag ggagttccta gggaaggcag gcttctgtcg cctcttcatc
     2281 cctgggtttg cagaaatggc agcccccctg taccctctca ccaaaccggg gactctgttt
     2341 aattggggcc cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc
     2401 ccagccctgg ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag
     2461 ggctacgcca aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac
     2521 ctgtccaaaa agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca
     2581 gccattgccg tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt
     2641 ctggcccccc atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac
     2701 gcccggatga ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg
     2761 gtggtagccc tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac
     2821 tgcctttctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     2881 aggaaagtcg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag
     2941 gagaaccctg gacctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3001 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3061 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3121 gtgccctggc ccaccctcgt gaccaccctg acctatggag tgcagtgctt cagccgctac
     3181 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3241 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3301 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3361 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     3421 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3481 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3541 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     3601 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3661 gagctgtaca agtaaaaacg ttgttgtttg gggttgaatt actcttgatc agataatcaa
     3721 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3781 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3841 ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc
     3901 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3961 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct ttatttgtga
     4021 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     4081 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     4141 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4201 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4261 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4321 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4381 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     4441 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4501 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4561 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4621 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4681 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     4741 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4801 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4861 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4921 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4981 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5041 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5101 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5161 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5221 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5281 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5341 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5401 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5461 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5521 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5581 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5641 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5701 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5761 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5821 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5881 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5941 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6001 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6061 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6121 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6181 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6241 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6301 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6361 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6421 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6481 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6541 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6601 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6661 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6721 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6781 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6841 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcc
//
