LOCUS       sequence-358233-        5781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector carrying a guide RNA targeting the human SMAD4 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 interspersed loops (SMAD4)
FEATURES             Location/Qualifiers
     source          1..5781
                     /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        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        819..1132
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1051..1071
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(1144..1168)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1183..1562
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1563..1766
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1717..1737
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1763..1787
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1878..1887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1884..1886,1887..1889,1890..2600)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1884..1886,1887..1889,1890..2600)
                     /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(1929..1950)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2190..2209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2537..2558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2679..2698)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2723..2771
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   2986..3126
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2986..3115
                     /label=AAV2 ITR
     primer_bind     complement(3166..3182)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3166..3182)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3179..3201)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3190..3206
                     /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(3214..3220,3221..3238,3239..3244))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3259..3280
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3568..4156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3648..3667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4327..5142)
                     /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(4327..5142)
                     /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     5050..5069
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5143..5247)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5315..5333
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5371..5393)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5493..5512
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5706..5728
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5721..5737
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5762..5778
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtggagg agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc catcagtcgc
      421 cggtcccaag cccggataaa atgggagggg gcgggaaacc gcctaaccat gccgactgat
      481 ggcagaaaaa aaaaattgta gtccaccatc ctgataaggt taacccgccc aacggtaaaa
      541 gaggagagtc taaaggttgt gccagtgcaa tcggcatggt atgaagtact tcgtccagga
      601 ggaccagggc ccggtgtaag acggtttcaa tccagcaagc acattctttg atgctctgag
      661 aagggtaatc cggtccccag cctttcacaa aactcaaaaa aaaagctgcc atcagtcggc
      721 gtggactgta gaacactgcc aatgccggtc ccaagcccgg ataaaagtgg agggtacagt
      781 ccacgctttt ttcgtactga gtcgcccagt ctcagataga tccgacgccg ccatctctag
      841 gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg
      901 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
      961 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag
     1021 atacaaatac taaattatta ttttaaaaaa cagcacaaaa ggaaactcac cctaactgta
     1081 aagtaattgt gtgttttgag actataaata tcccttggag aaaagccttg tttggattcc
     1141 tagacccagc tttcttgtac aaagttggca ttaatacgcg ttgacattga ttattgacta
     1201 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     1261 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     1321 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     1381 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     1441 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1501 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1561 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     1621 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1681 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1741 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     1801 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cggactctag
     1861 aggatcgaac ccttaaggcc accatggtga gcaagggcga ggagctgttc accggggtgg
     1921 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1981 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2041 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2101 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2161 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2221 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2281 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2341 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2401 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2461 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2521 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2581 gcatggacga gctgtacaag tactcagatc tcgagctcaa gtagcttaag ggttcgatcc
     2641 ctactggtta gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata
     2701 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg
     2761 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     2821 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     2881 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga
     2941 tctgcgctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     3001 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3061 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     3121 tgcaggcttg gatcccaatg gcgcgccgag cttggctcga gcatggtcat agctgtttcc
     3181 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3241 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3301 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3361 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3421 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3481 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3541 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3661 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3721 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3781 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3841 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3961 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4021 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4081 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4141 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4201 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4261 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4321 tgacagttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4381 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     4441 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     4501 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     4561 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     4621 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     4681 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     4741 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4801 aggatattct tctaatacct ggaatgctgt tttcccaggg atcgcagtgg tgagtaacca
     4861 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4921 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4981 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     5041 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     5101 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     5161 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5221 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     5281 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     5341 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5401 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5461 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     5521 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     5581 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     5641 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     5701 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgacgcgcc attgggatgt
     5761 tgtaaaacga cggccagtga a
//
