LOCUS       sequence-337169-        5779 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 5779)
  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 5779)
  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 (GAPDH)
FEATURES             Location/Qualifiers
     source          1..5779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..32
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   36..176
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   47..176
                     /label=AAV2 ITR
     promoter        195..443
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     195..215
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     366..385
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        852..1165
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1084..1104
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(1177..1201)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1216..1595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1596..1799
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1750..1770
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1796..1820
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1911..1920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1917..1919,1920..1922,1923..2633)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1917..1919,1920..1922,1923..2633)
                     /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(1962..1983)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2223..2242)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2570..2591
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2712..2731)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2756..2804
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   3019..3159
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3019..3148
                     /label=AAV2 ITR
     primer_bind     complement(3199..3215)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3199..3215)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3212..3234)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3223..3239
                     /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(3247..3253,3254..3271,3272..3277))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3292..3313
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3601..4189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3681..3700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4360..5175)
                     /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(4360..5175)
                     /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     5083..5102
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5176..5280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5348..5366
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5404..5426)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5526..5545
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5739..5761
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5754..5770
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgccattggg atgttgtaaa acgacggcca gtgaacctgc aggcagctgc gcgctcgctc
       61 gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc
      121 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg
      181 ccgcacgcgt ggaggagggc ctatttccca tgattccttc atatttgcat atacgataca
      241 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
      301 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      361 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      421 tcttgtggaa aggacgaaac accgccatca gtcgccggtc ccaagcccgg ataaaatggg
      481 agggggcggg aaaccgccta accatgccga ctgatgcaga aaaaaaaaag gccatccaca
      541 gtcttctggg tggcagtgat ggcatggact gtggtcatga gtccttccac gataccaaag
      601 ttgtcacgga tgaccttggc caggggtgcc aagcagttgg tggtgcagga ggcattgctg
      661 atgatcttga ggctgttgtc atacttctca tggttcacac ccatgacgaa catgggggca
      721 tcagcagaga aaaaaaagct gccatcagtc ggcgtggact gtagaacact gccaatgccg
      781 gtcccaagcc cggataaaag tggagggtac agtccacgct tttttcgtac tgagtcgccc
      841 agtctcagat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact
      901 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta
      961 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac
     1021 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa
     1081 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa
     1141 atatcccttg gagaaaagcc ttgtttggat tcctagaccc agctttcttg tacaaagttg
     1201 gcattaatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     1261 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1321 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1381 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1441 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1501 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1561 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     1621 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     1681 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     1741 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     1801 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     1861 gacaccggga ccgatccagc ctccggactc tagaggatcg aacccttaag gccaccatgg
     1921 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1981 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2041 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2101 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2161 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2221 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2281 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2341 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2401 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2461 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2521 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2581 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtactcag
     2641 atctcgagct caagtagctt aagggttcga tccctactgg ttagtaatga gtttaaacgg
     2701 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     2761 ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg
     2821 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     2881 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     2941 caacgtcggg gcggcaggcc ctgccatagc agatctgcgc tgattttgta ggtaaccacg
     3001 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3061 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3121 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggc ttggatccca atggcgcgcc
     3181 gagcttggct cgagcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3241 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3301 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3361 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3421 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3481 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3541 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3601 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3661 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3721 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3781 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3841 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3901 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3961 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4021 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4081 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4141 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4201 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4261 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4321 atcaatctaa agtatatatg agtaaacttg gtctgacagt tagaaaaact catcgagcat
     4381 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     4441 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     4501 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     4561 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     4621 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     4681 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     4741 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     4801 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     4861 tgttttccca gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     4921 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     4981 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     5041 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     5101 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     5161 ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5281 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     5341 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     5401 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5461 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     5521 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     5581 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     5641 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     5701 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     5761 gacggccagt gaattgacg
//
