LOCUS       sequence-374556-        7483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing the C-terminal of PE_CO-Mini using Rma intein and Cas9 
            673-674 split site, and pegRNA for PCSK9 +1A insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7483)
  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 7483)
  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-C
FEATURES             Location/Qualifiers
     source          1..7483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        159..407
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     159..179
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     330..349
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        429..504
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             845..865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4652..4672
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4682..4737
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4746..4886
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4746..4875
                     /label=AAV2 ITR
     rep_origin      4961..5416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5048..5067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5258..5279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5433..5452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5552..5574
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5612..5630)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5698..5802
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5803..5871,5872..6663)
                     /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(6021..6040)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6834..7422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7323..7342
                     /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 gcggccgcga cattgattat tgactagtga gggcctattt cccatgattc
      181 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
      241 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      301 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      361 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgcc cataccttgg
      421 agcaacgggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      481 gaaaaagtgg caccgagtcg gtgcaccttc cgccgattgc tccaaggttt tttactagta
      541 tggaggcggt actatgtaga tgagaattca ggagcaaact gggaaaagca actgcttcca
      601 aatatttgtg atttttacag tgtagttttg gaaaaactct tagcctacca attcttctaa
      661 gtgttttaaa atgtgggagc cagtacacat gaagttatag agtgttttaa tgaggcttaa
      721 atatttaccg taactatgaa atgctacgca tatcatgctg ttcaggctcc gtggccacgc
      781 aactcatact gaccggtgag ccgccaccat gaaacggaca gccgacggaa gcgagttcga
      841 gtcaccaaag aagaagcgga aagtcgcggc ggcgtgcccg gaactgcgtc agctggcgca
      901 gagcgatgtg tattgggatc cgattgtgag cattgaaccg gatggcgtgg aagaagtgtt
      961 tgatctgacc gtgccgggcc cgcataactt tgtggcgaac gatattattg cgcataactc
     1021 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     1081 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     1141 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     1201 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     1261 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     1321 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     1381 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     1441 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     1501 ctacgatgtg gacgctatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     1561 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     1621 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     1681 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     1741 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     1801 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     1861 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     1921 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     1981 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     2041 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     2101 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     2161 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     2221 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     2281 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     2341 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     2401 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     2461 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     2521 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     2581 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     2641 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     2701 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     2761 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     2821 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     2881 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     2941 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     3001 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     3061 gtacgagaca cggatcgacc tgtctcagct gggaggtgac tctggaggat ctagcggagg
     3121 atcctctggc agcgagacac caggaacaag cgagtcagca acaccagaga gcagtggcgg
     3181 cagcagcggc ggcagcagcc tgggttctac atggctgagc gacttccctc aggcctgggc
     3241 tgaaaccggc ggcatgggcc tggctgtgcg gcaggctcct ctgatcatcc ctctgaaggc
     3301 cacctccacc cctgtgtcca tcaagcagta ccccatgagc caggaggcca gactgggcat
     3361 caagccccac atccagcggc tgctcgacca gggaatcctg gtcccctgcc agagcccctg
     3421 gaacacccca cttttacctg tgaaaaagcc cggcactaac gactaccggc ctgtgcagga
     3481 cctgagagaa gtgaacaagc gggttgagga catccaccct acagtgccta acccctacaa
     3541 cctgctgtct ggcctgccac cgagccacca gtggtacacc gtgctggacc tgaaagacgc
     3601 cttcttctgt ctgagactcc atcctaccag ccagccactg ttcgccttcg agtggcggga
     3661 ccctgagatg ggcatcagcg gccagctgac atggacccgg ctgccacagg gcttcaagaa
     3721 cagccctaca ctgttcaacg aggccctgca cagagatctg gctgacttca gaatccagca
     3781 tcctgacctg atcctgctgc agtacgtgga cgacttgctg cttgccgcta catctgaact
     3841 ggattgccag caaggcacca gagctctgct ccagaccctg ggcaacctgg ggtatagggc
     3901 cagcgccaag aaggcccaga tctgccagaa gcaagtgaag tacctgggat acctgctgaa
     3961 ggaaggccag agatggctga ccgaggcgcg gaaggaaaca gtgatgggcc agcctacccc
     4021 taagaccccc agacagctgc gagaattcct gggcaaggct ggcttttgca gactgttcat
     4081 ccccggcttc gctgagatgg ccgctcctct gtacccactg accaagcccg gtaccctgtt
     4141 caattggggc cccgatcagc agaaagccta ccaggagatt aagcaggccc tgctgaccgc
     4201 ccctgccctc gggctgcctg atctgacaaa acctttcgag ctgtttgtcg acgagaagca
     4261 gggctacgcc aagggcgtgc tgacacagaa actgggacct tggagaagac ctgtggctta
     4321 tctcagcaag aagctggacc ccgtggccgc cggatggcct ccatgtctga ggatggtcgc
     4381 cgctatcgcc gtgctgacca aggacgccgg caaactgacc atgggacaac ctctggtgat
     4441 cctggcccct cacgccgtcg aggccctggt gaagcagcct cctgacagat ggctgagcaa
     4501 cgccagaatg acccactacc aggccctgct gctggatacc gatagagtgc agttcggccc
     4561 tgtggtggct ctgaatcctg ccaccctgct cccacttccc gaggaaggct ctggcggctc
     4621 aaaaagaacc gccgacggca gcgaattcga gcccaagaag aagaggaaag tctaatctag
     4681 aaataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcagcg
     4741 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4801 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4861 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4921 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4981 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     5041 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5101 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5161 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5221 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5281 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5341 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5401 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5461 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5521 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5581 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5641 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5701 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5761 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5821 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5881 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5941 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6001 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6061 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6121 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6181 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6241 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6301 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6361 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6421 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6481 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6541 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6601 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6661 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6721 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6961 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7141 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7441 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
