LOCUS       sequence-368872-        7493 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 7493)
  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 7493)
  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..7493
                     /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"
     CDS             855..875
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4662..4682
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4692..4747
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4756..4896
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4756..4885
                     /label=AAV2 ITR
     rep_origin      4971..5426
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5058..5077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5268..5289
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5443..5462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5562..5584
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5622..5640)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5708..5812
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5813..5881,5882..6673)
                     /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(6031..6050)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6844..7432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7333..7352
                     /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 ttcagagcta tgctggaaac agcatagcaa gttgaaataa ggctagtccg
      481 ttatcaactt gaaaaagtgg caccgagtcg gtgcaccttc cgccgattgc tccaaggttt
      541 tttactagta tggaggcggt actatgtaga tgagaattca ggagcaaact gggaaaagca
      601 actgcttcca aatatttgtg atttttacag tgtagttttg gaaaaactct tagcctacca
      661 attcttctaa gtgttttaaa atgtgggagc cagtacacat gaagttatag agtgttttaa
      721 tgaggcttaa atatttaccg taactatgaa atgctacgca tatcatgctg ttcaggctcc
      781 gtggccacgc aactcatact gaccggtgag ccgccaccat gaaacggaca gccgacggaa
      841 gcgagttcga gtcaccaaag aagaagcgga aagtcgcggc ggcgtgcccg gaactgcgtc
      901 agctggcgca gagcgatgtg tattgggatc cgattgtgag cattgaaccg gatggcgtgg
      961 aagaagtgtt tgatctgacc gtgccgggcc cgcataactt tgtggcgaac gatattattg
     1021 cgcataactc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     1081 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     1141 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     1201 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     1261 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     1321 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     1381 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     1441 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     1501 ggctgtccga ctacgatgtg gacgctatcg tgcctcagag ctttctgaag gacgactcca
     1561 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     1621 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     1681 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     1741 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     1801 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     1861 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     1921 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     1981 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     2041 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     2101 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     2161 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     2221 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     2281 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     2341 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     2401 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     2461 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     2521 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     2581 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     2641 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     2701 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     2761 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     2821 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     2881 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     2941 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     3001 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     3061 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac tctggaggat
     3121 ctagcggagg atcctctggc agcgagacac caggaacaag cgagtcagca acaccagaga
     3181 gcagtggcgg cagcagcggc ggcagcagcc tgggttctac atggctgagc gacttccctc
     3241 aggcctgggc tgaaaccggc ggcatgggcc tggctgtgcg gcaggctcct ctgatcatcc
     3301 ctctgaaggc cacctccacc cctgtgtcca tcaagcagta ccccatgagc caggaggcca
     3361 gactgggcat caagccccac atccagcggc tgctcgacca gggaatcctg gtcccctgcc
     3421 agagcccctg gaacacccca cttttacctg tgaaaaagcc cggcactaac gactaccggc
     3481 ctgtgcagga cctgagagaa gtgaacaagc gggttgagga catccaccct acagtgccta
     3541 acccctacaa cctgctgtct ggcctgccac cgagccacca gtggtacacc gtgctggacc
     3601 tgaaagacgc cttcttctgt ctgagactcc atcctaccag ccagccactg ttcgccttcg
     3661 agtggcggga ccctgagatg ggcatcagcg gccagctgac atggacccgg ctgccacagg
     3721 gcttcaagaa cagccctaca ctgttcaacg aggccctgca cagagatctg gctgacttca
     3781 gaatccagca tcctgacctg atcctgctgc agtacgtgga cgacttgctg cttgccgcta
     3841 catctgaact ggattgccag caaggcacca gagctctgct ccagaccctg ggcaacctgg
     3901 ggtatagggc cagcgccaag aaggcccaga tctgccagaa gcaagtgaag tacctgggat
     3961 acctgctgaa ggaaggccag agatggctga ccgaggcgcg gaaggaaaca gtgatgggcc
     4021 agcctacccc taagaccccc agacagctgc gagaattcct gggcaaggct ggcttttgca
     4081 gactgttcat ccccggcttc gctgagatgg ccgctcctct gtacccactg accaagcccg
     4141 gtaccctgtt caattggggc cccgatcagc agaaagccta ccaggagatt aagcaggccc
     4201 tgctgaccgc ccctgccctc gggctgcctg atctgacaaa acctttcgag ctgtttgtcg
     4261 acgagaagca gggctacgcc aagggcgtgc tgacacagaa actgggacct tggagaagac
     4321 ctgtggctta tctcagcaag aagctggacc ccgtggccgc cggatggcct ccatgtctga
     4381 ggatggtcgc cgctatcgcc gtgctgacca aggacgccgg caaactgacc atgggacaac
     4441 ctctggtgat cctggcccct cacgccgtcg aggccctggt gaagcagcct cctgacagat
     4501 ggctgagcaa cgccagaatg acccactacc aggccctgct gctggatacc gatagagtgc
     4561 agttcggccc tgtggtggct ctgaatcctg ccaccctgct cccacttccc gaggaaggct
     4621 ctggcggctc aaaaagaacc gccgacggca gcgaattcga gcccaagaag aagaggaaag
     4681 tctaatctag aaataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     4741 tctctcagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4801 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4861 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4921 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4981 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5041 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5101 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5161 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     5221 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5281 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     5341 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5401 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5461 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5521 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5581 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5641 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5701 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5761 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5821 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5881 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5941 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6001 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6061 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6121 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6181 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6241 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6301 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6361 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6421 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6481 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6541 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6601 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6661 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6721 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6781 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6841 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6901 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6961 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     7021 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7081 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7141 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7201 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7261 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7321 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7381 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7441 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
