LOCUS       sequence-314789-        4956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. pyogenes pegRNA for C to A mutation at the KCNA1 site of human 
            cells using prime editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4956)
  AUTHORS   Liu Y, Li X, He S, Huang S, Li C, Chen Y, Liu Z, Huang X, Wang X
  TITLE     Efficient generation of mouse models with the prime editing system.
  JOURNAL   Cell Discov. 2020 Apr 28;6:27. doi: 10.1038/s41421-020-0165-z. 
            eCollection 2020.
  PUBMED    32351707
REFERENCE   2  (bases 1 to 4956)
  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: "Cell 
            Discov."; date: "2020-04-28"; volume: "6:27. doi"; pages: " 
            10.1038/s41421-020-0165-z. eCollection 2020"
COMMENT     Sequence Label: pGL3-U6-pegRNA_KCNA1-EGFP
FEATURES             Location/Qualifiers
     source          1..4956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    428..545
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        594..1104
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1023..1041
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(1113..1115,1116..1118,1119..1832)
                     /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(1113..1115,1116..1118,1119..1829)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1158..1179)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1419..1438)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1766..1787
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1955..2076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1992..2011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2046..2065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2217..2234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2388..2976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2468..2487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3147..3938,3939..4007))
                     /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     3770..3789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4008..4112)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4139..4594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4226..4245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4436..4457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4725..4773
                     /note="synthetic polyadenylation signal"
     misc_feature    4787..4878
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ccacctccct ggccatcctc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaagc ggatgaccct
      361 aaggatggcc agggagtttt tttaaagaat tctcgacctc gagacaaatg gcagtattca
      421 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
      481 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
      541 attttcgggt ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg
      601 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
      661 ggttccggga aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc
      721 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
      781 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
      841 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
      901 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
      961 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     1021 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     1081 cgaatcaccg acctctctcc ccagggggat ccatggtgag caagggcgag gagctgttca
     1141 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1201 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1261 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1321 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1381 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1441 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1501 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1561 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1621 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1681 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     1741 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1801 tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag
     1861 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     1921 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     1981 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2041 tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg accgatgccc ttgagagcct
     2101 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     2161 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg
     2221 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2281 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca
     3301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4141 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4201 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4261 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4321 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4381 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4441 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4501 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4561 gcgaatttta acaaaatatt aacgcttaca atttgccatt cgccattcag gctgcgcaac
     4621 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga
     4681 taagtaagta atattaaggt acgggaggta cttggagcgg ccgcaataaa atatctttat
     4741 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
     4801 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccacagt gcaagtgcag
     4861 gtgccagaac atttctctat cgataggtac cgattagtga acggatctcg acggtatcga
     4921 tcacgagact agcctcgagc ggccgccccc ttcacc
//
