LOCUS       sequence-342981-        6551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses prime editing guide RNA and guide RNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  AUTHORS   Jang H, Jo DH, Cho CS, Shin JH, Seo JH, Yu G, Gopalappa R, Kim D, 
            Cho SR, Kim JH, Kim HH
  TITLE     Application of prime editing to the correction of mutations and 
            phenotypes in adult mice with liver and eye diseases.
  JOURNAL   Nat Biomed Eng. 2021 Aug 26. pii: 10.1038/s41551-021-00788-9. doi: 
            10.1038/s41551-021-00788-9.
  PUBMED    34446856
REFERENCE   2  (bases 1 to 6551)
  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: "Nat Biomed 
            Eng. 2021 Aug 26. pii: 10.1038/s41551-021-00788-9. doi: 
            10.1038/s41551-021-00788-9."
COMMENT     Sequence Label: U6-pegRNA-H1-nick sgRNA-mCherry
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     673..692
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        782..857
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        865..1244
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1246..1523
                     /label=chicken beta-actin promoter
     intron          1524..2535
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2459..2481
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      2544..2553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2550..3260
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2695..2713)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2963..2982
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3298..3774
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3497..3516)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3814..3954
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3814..3943
                     /label=AAV2 ITR
     rep_origin      4029..4484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4116..4135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4326..4347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4501..4520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4620..4642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4680..4698)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4766..4870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4871..4939,4940..5731)
                     /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(5089..5108)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5902..6490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6391..6410
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgcct ttcagtttct
      421 gggtgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      481 aagtgggacc gagtcggtcc agtaccaccc atccagaaac tgaaagtttt tttgtttgaa
      541 cgctgacgtc atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac
      601 ccagcgcgcg tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc
      661 aatatttgca tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat
      721 ttgggaatct tataagttct gtatgaggac cacagatccc cgatgagtac gtccatgttg
      781 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      841 tggcaccgag tcggtgcttt ttttgacatt gattattgac tagttattaa tagtaatcaa
      901 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      961 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1021 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1081 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1141 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1201 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     1261 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     1321 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
     1381 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     1441 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     1501 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgtcgcg ccttcgcccc gtgccccgct
     1561 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1621 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggctcg
     1681 tttcttttct gtggctgcgt gaaagcctta aagggctccg ggagggccct ttgtgcgggg
     1741 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggcccg
     1801 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcgtgt
     1861 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc gggggggctg cgaggggaac
     1921 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc
     1981 gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg
     2041 tgcggggctc cgtgcggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca
     2101 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg
     2161 cggcggcccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     2221 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctggcg gagccgaaat
     2281 ctgggaggcg ccgccgcacc ccctctagcg ggcgcgggcg aagcggtgcg gcgccggcag
     2341 gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccatct
     2401 ccagcctcgg ggctgccgca gggggacggc tgccttcggg ggggacgggg cagggcgggg
     2461 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
     2521 ttctttttcc tacagtcgac gtcgccacca tggtgagcaa gggcgaggag gataacatgg
     2581 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     2641 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     2701 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     2761 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     2821 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2881 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2941 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     3001 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     3061 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     3121 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     3181 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     3241 tggacgagct gtacaagtga aagcttatcg ataccgagcg ctgctcgaga gatctacggg
     3301 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     3361 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     3421 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     3481 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     3541 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     3601 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     3661 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     3721 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     3781 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     3841 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3901 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3961 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4021 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4081 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4141 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4201 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4261 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4321 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt
     4381 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4441 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4501 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4561 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4621 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4681 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4801 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4861 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4921 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4981 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5041 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5101 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5161 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5221 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5281 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5341 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5401 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5461 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5521 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5581 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5641 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5701 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5761 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5821 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5881 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5941 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6001 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     6061 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6121 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6181 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6241 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6301 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6361 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6421 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6481 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6541 tgctcacatg t
//
