LOCUS       sequence-374544-        6425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing the N-terminal of PE_CO-Mini using Rma intein and Cas9 
            673-674 split site, ngRNA and pegRNA for PCSK9 +1A insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6425)
  AUTHORS   Gao Z, Ravendran S, Mikkelsen NS, Haldrup J, Cai H, Ding X, Paludan 
            SR, Thomsen MK, Mikkelsen JG, Bak RO
  TITLE     A truncated reverse transcriptase enhances prime editing by split 
            AAV vectors.
  JOURNAL   Mol Ther. 2022 Jul 8. pii: S1525-0016(22)00422-1. doi: 
            10.1016/j.ymthe.2022.07.001.
  PUBMED    35808824
REFERENCE   2  (bases 1 to 6425)
  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: "Mol Ther. 
            2022 Jul 8. pii: S1525-0016(22)00422-1. doi: 
            10.1016/j.ymthe.2022.07.001."
COMMENT     Sequence Label: PE_CO-Mini-N
FEATURES             Location/Qualifiers
     source          1..6425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(151..226)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(248..496)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     CDS             808..828
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             829..2544
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             3196..3216
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3226..3281
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     promoter        3288..3536
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   3688..3828
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3688..3817
                     /label=AAV2 ITR
     rep_origin      3903..4358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3990..4009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4200..4221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4375..4394)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4494..4516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4554..4572)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4640..4744
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4745..4813,4814..5605)
                     /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(4963..4982)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5776..6364
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6265..6284
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcaa gcttaaaaaa gcaccgactc ggtgccactt tttcaagttg
      181 ataacggact agccttattt taacttgcta tttctagctc taaaactgga gcaatcggcg
      241 gaaggtcggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
      301 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
      361 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
      421 aattaattcc aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
      481 tgggaaatag gccctcctcg agatggaggc ggtactatgt agatgagaat tcaggagcaa
      541 actgggaaaa gcaactgctt ccaaatattt gtgattttta cagtgtagtt ttggaaaaac
      601 tcttagccta ccaattcttc taagtgtttt aaaatgtggg agccagtaca catgaagtta
      661 tagagtgttt taatgaggct taaatattta ccgtaactat gaaatgctac gcatatcatg
      721 ctgttcaggc tccgtggcca cgcaactcat actgaccggt gagccgccac catgaaacgg
      781 acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcga caagaagtac
      841 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
      901 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
      961 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1021 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1081 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1141 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1201 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1261 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1321 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1381 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     1441 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     1501 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     1561 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     1621 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     1681 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     1741 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     1801 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     1861 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     1921 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     1981 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     2041 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2101 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2161 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2221 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2281 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2341 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     2401 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     2461 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     2521 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     2581 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     2641 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     2701 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     2761 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     2821 ctgatcaacg gcatccggga caagcagtgt ctggctggcg atactctcat taccctggcc
     2881 gatggacgac gagtgcctat tagagaactg gtgtcacagc agaatttttc cgtgtgggct
     2941 ctgaatcctc agacttaccg cctggagagg gctagagtga gtagagcttt ctgtaccggc
     3001 atcaaacctg tgtaccgcct caccactaga ctggggagat ccattagggc cactgccaac
     3061 caccgatttc tcacacctca gggctggaaa cgagtcgatg aactccagcc tggagattac
     3121 ctggctctgc ctaggagaat ccctactgcc tccagcggag gcagtaaaag aacagcagac
     3181 gggagtgagt ttgagcccaa gaaaaagaga aaggtgtaat ctagaaataa aggaaattta
     3241 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc aggatccgag ggcctatttc
     3301 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     3361 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     3421 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     3481 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgccc
     3541 ataccttgga gcaacgggtt tcagagctat gctggaaaca gcatagcaag ttgaaataag
     3601 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcaccttcc gccgattgct
     3661 ccaaggtttt tttgaattcg cggccgcagg aacccctagt gatggagttg gccactccct
     3721 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     3781 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     3841 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     3901 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3961 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4021 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4081 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4141 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4201 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     4261 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     4321 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     4381 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     4441 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4501 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     4561 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4621 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4681 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4741 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4801 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4861 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4921 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4981 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5041 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5101 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5161 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5221 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5281 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5341 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5401 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5461 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5521 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5581 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5641 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5701 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5761 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5821 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5881 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5941 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6001 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6061 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6121 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6181 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6241 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6301 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6361 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6421 catgt
//
