LOCUS Exported 6551 bp ds-DNA circular SYN 17-SEP-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 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 Sep 17, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6551 /organism="synthetic DNA construct" /mol_type="other DNA" 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..396 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 4871..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 //