LOCUS       Exported                6503 bp ds-DNA     circular SYN 13-DEC-2021
DEFINITION  Expresses prime editing guide RNA and guide RNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    U6-pegRNA-H1-nick sgRNA-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6503)
  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 6503)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6503
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     intron          123..1139
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1063..1085
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1148..1157
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1154..1864
                     /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(1299..1317)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1567..1586
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1902..2378
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2101..2120)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2418..2558
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2418..2547
                     /label=AAV2 ITR
     rep_origin      2633..3088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2720..2739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2930..2951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3105..3124)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3224..3246
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3284..3302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3370..3474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3475..4335
                     /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(3693..3712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4506..5094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4995..5014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5156..5285
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5300..5540
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     5300..5320
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5471..5490
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5569..5644
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5817..5836
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        5926..6001
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        6009..6388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
       61 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      121 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      181 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      241 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      301 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      361 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      421 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
      481 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
      541 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
      601 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
      661 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
      721 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
      781 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
      841 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
      901 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
      961 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1021 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1081 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagt
     1141 cgacgtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     1201 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     1261 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1321 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     1381 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     1441 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1501 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1561 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1621 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1681 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1741 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1801 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1861 gtgaaagctt atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     1921 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     1981 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     2041 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     2101 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     2161 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     2221 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     2281 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     2341 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     2401 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     2461 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     2521 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     2581 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     2641 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2701 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2761 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2821 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     2881 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2941 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     3001 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3061 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     3121 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3181 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3241 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     3301 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3361 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3421 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3481 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3541 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3601 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3661 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     3721 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3781 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3841 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3901 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3961 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4021 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4081 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4141 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4201 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4261 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4321 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4381 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4441 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4501 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4561 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4621 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4681 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4741 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4801 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4861 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4921 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4981 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5041 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5101 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc
     5161 aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg
     5221 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
     5281 ttcctgcggc cgcacgcgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     5341 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     5401 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     5461 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     5521 atatatcttg tggaaaggac gaaacaccgt gcctttcagt ttctgggtgt tttagagcta
     5581 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg gaccgagtcg
     5641 gtccagtacc acccatccag aaactgaaag tttttttgtt tgaacgctga cgtcatcaac
     5701 ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc
     5761 ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc
     5821 tatgtgttct gggaaatcac cataaacgtg aaatgtcttt ggatttggga atcttataag
     5881 ttctgtatga ggaccacaga tccccgatga gtacgtccat gttgggtttt agagctagaa
     5941 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     6001 ctttttttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     6061 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     6121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     6181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     6241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     6301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     6361 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     6421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     6481 tgcagcgatg ggggcggggg ggg
//
