LOCUS       sequence-337245-        5781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector carrying a guide RNA targeting the human GAPDH mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5781)
  AUTHORS   Katrekar D, Yen J, Xiang Y, Saha A, Meluzzi D, Savva Y, Mali P
  TITLE     Efficient in vitro and in vivo RNA editing via recruitment of 
            endogenous ADARs using circular guide RNAs.
  JOURNAL   Nat Biotechnol. 2022 Feb 10. pii: 10.1038/s41587-021-01171-4. doi: 
            10.1038/s41587-021-01171-4.
  PUBMED    35145312
REFERENCE   2  (bases 1 to 5781)
  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 
            Biotechnol. 2022 Feb 10. pii: 10.1038/s41587-021-01171-4. doi: 
            10.1038/s41587-021-01171-4."
COMMENT     Sequence Label: Circular 200,100 with 8 bp bulge loops (GAPDH)
FEATURES             Location/Qualifiers
     source          1..5781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   63..203
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   63..192
                     /label=AAV2 ITR
     primer_bind     complement(243..259)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(243..259)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(256..278)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    267..283
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(291..297,298..315,316..321))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    336..357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(645..1233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(725..744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1404..2219)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1404..2219)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2127..2146
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2220..2324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2392..2410
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2448..2470)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2570..2589
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2783..2805
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2798..2814
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2839..2855
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2859..2999
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2870..2999
                     /label=AAV2 ITR
     promoter        3018..3266
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3018..3038
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3189..3208
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3677..3990
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     3909..3929
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(4002..4026)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        4041..4420
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4421..4624
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4575..4595
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4621..4645
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      4736..4745
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4742..4744,4745..4747,4748..5458)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4742..4744,4745..4747,4748..5458)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4787..4808)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5048..5067)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5395..5416
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5537..5556)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5581..5629
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ggccctgcca tagcagatct gcgctgattt tgtaggtaac cacgtgcgga ccgagcggcc
       61 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
      121 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
      181 gcgagcgcgc agctgcctgc aggcttggat cccaatggcg cgccgagctt ggctcgagca
      241 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      301 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      361 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
      421 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
      481 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      541 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      601 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      661 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      721 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      781 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      841 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      901 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      961 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1021 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1081 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1141 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1201 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1261 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1321 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1381 tatgagtaaa cttggtctga cagttagaaa aactcatcga gcatcaaatg aaactgcaat
     1441 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1501 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1561 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1621 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1681 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1741 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     1801 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1861 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccagggatc
     1921 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1981 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2041 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2101 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2161 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2221 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2281 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2341 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2401 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2461 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2521 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     2581 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     2641 aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2701 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     2761 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg
     2821 acgcgccatt gggatgttgt aaaacgacgg ccagtgaacc tgcaggcagc tgcgcgctcg
     2881 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
     2941 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg
     3001 cggccgcacg cgtggaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     3061 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     3121 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     3181 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     3241 atatcttgtg gaaaggacga aacaccgcca tcagtcgccg gtcccaagcc cggataaaat
     3301 gggagggggc gggaaaccgc ctaaccatgc cgactgatgg cagaaaaaaa aaaggccatc
     3361 cacagtcttc tgggtggcag tgatggcatg gactgtggtc atgagtcctt ccacgatacg
     3421 ttcgttgtca tggatgacct tggccagggg tgccaagcac aacgtggtgc aggaggcatt
     3481 gctgatgatc ttgaggctgt tgtcatactt ctcatggttc acacccatga cgaacatggg
     3541 ggcatcagca gagaaaaaaa aagctgccat cagtcggcgt ggactgtaga acactgccaa
     3601 tgccggtccc aagcccggat aaaagtggag ggtacagtcc acgctttttt cgtactgagt
     3661 cgcccagtct cagatagatc cgacgccgcc atctctaggc ccgcgccggc cccctcgcac
     3721 agacttgtgg gagaagctcg gctactcccc tgccccggtt aatttgcata taatatttcc
     3781 tagtaactat agaggcttaa tgtgcgataa aagacagata atctgttctt tttaatacta
     3841 gctacatttt acatgatagg cttggatttc tataagagat acaaatacta aattattatt
     3901 ttaaaaaaca gcacaaaagg aaactcaccc taactgtaaa gtaattgtgt gttttgagac
     3961 tataaatatc ccttggagaa aagccttgtt tggattccta gacccagctt tcttgtacaa
     4021 agttggcatt aatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
     4081 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     4141 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     4201 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     4261 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     4321 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     4381 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     4441 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     4501 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     4561 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     4621 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
     4681 tagaagacac cgggaccgat ccagcctccg gactctagag gatcgaaccc ttaaggccac
     4741 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4801 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     4861 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4921 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4981 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     5041 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     5101 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     5161 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     5221 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     5281 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     5341 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     5401 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     5461 ctcagatctc gagctcaagt agcttaaggg ttcgatccct actggttagt aatgagttta
     5521 aacgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5581 cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg
     5641 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5701 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5761 gcagccaacg tcggggcggc a
//
