LOCUS       sequence-337172-        6868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector carrying 2 copies of a guide RNA targeting the mouse IDUA
            mRNA, expressed from human and mouse U6 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6868)
  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 6868)
  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 (mIDUA)
FEATURES             Location/Qualifiers
     source          1..6868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          561..693
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     676..695
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      716..725
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             722..1432
                     /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(867..885)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1135..1154
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1502..1522)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1920..1931)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1998..2015)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2004..2211
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2213..2461
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2213..2233
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2384..2403
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2868..3181
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     3100..3120
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     repeat_region   3648..3777
                     /label=AAV2 ITR
     primer_bind     complement(3796..3813)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3796..3812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3805..3827)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3954..4409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4041..4060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4251..4272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4435..4539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4540..4608,4609..5400)
                     /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(4758..4777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5571..6159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6060..6079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6447..6468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6483..6488,6489..6506,6507..6513)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6521..6537
                     /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)."
     primer_bind     6526..6548
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6545..6561
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6545..6561
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6599..6728
                     /label=AAV2 ITR
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc tgcagaagtt
      541 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
      601 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
      661 ttactgacat ccactttgcc tttctctcca caggtgtcca ggcggcccta gcgccgccac
      721 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
      781 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      841 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
      901 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
      961 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1021 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1081 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1141 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1201 cgctctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1261 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggag cctacaacgt
     1321 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     1381 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aaaccggagc
     1441 ttggatccaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     1501 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     1561 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     1621 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     1681 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     1741 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     1801 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     1861 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     1921 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     1981 cgcgtcttcg agatctgcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2041 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2101 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2161 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg acgagggcct
     2221 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     2281 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     2341 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     2401 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     2461 cgccatcagt cgccggtccc aagcccggat aaaatgggag ggggcgggaa accgcctaac
     2521 catgccgact gatggcagaa aaaaaaaact ccaggccgct gcggagcctt cagggtgatg
     2581 ggtgctggcc aggacaccca ctgtatgatt gctgtccaac acagccccag cctttgagac
     2641 ctctgcccag agttgttctc catccaacag ggccatgagc cccatgactg tgagtactgg
     2701 ctttcgcagc aactgcacgt ggggtgggtg agtattgttg acctggaaaa aaaaactgcc
     2761 atcagtcggc gtggactgta gaacactgcc aatgccggtc ccaagcccgg ataaaagtgg
     2821 agggtacagt ccacgctttt ttgtactgag tcgcccagtc tcagatagat ccgacgccgc
     2881 catctctagg cccgcgccgg ccccctcgca cagacttgtg ggagaagctc ggctactccc
     2941 ctgccccggt taatttgcat ataatatttc ctagtaacta tagaggctta atgtgcgata
     3001 aaagacagat aatctgttct ttttaatact agctacattt tacatgatag gcttggattt
     3061 ctataagaga tacaaatact aaattattat tttaaaaaac agcacaaaag gaaactcacc
     3121 ctaactgtaa agtaattgtg tgttttgaga ctataaatat cccttggaga aaagccttgt
     3181 ttgggccatc agtcgccggt cccaagcccg gataaaatgg gagggggcgg gaaaccgcct
     3241 aaccatgccg actgatggca gaaaaaaaaa actccaggcc gctgcggagc cttcagggtg
     3301 atgggtgctg gccaggacac ccactgtatg attgctgtcc aacacagccc cagcctttga
     3361 gacctctgcc cagagttgtt ctccatccaa cagggccatg agccccatga ctgtgagtac
     3421 tggctttcgc agcaactgca cgtggggtgg gtgagtattg ttgacctgga aaaaaaaact
     3481 gccatcagtc ggcgtggact gtagaacact gccaatgccg gtcccaagcc cggataaaag
     3541 tggagggtac agtccacgct tttttgttaa gggcgaattc ccgataatga tcttcctaga
     3601 gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     3661 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3721 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct
     3781 taattaacct aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     3841 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     3901 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     3961 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4021 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4081 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4141 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4201 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4261 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4321 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4381 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4441 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4501 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4561 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4621 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4681 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4741 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4801 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4861 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4921 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4981 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5041 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5101 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5161 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5221 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5281 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5341 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5401 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5461 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5521 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5581 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5641 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5701 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5761 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5821 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5881 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5941 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6001 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6061 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6121 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6181 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6241 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6301 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6361 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6421 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     6481 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6541 cacacaggaa acagctatga ccatgattac gccagattta attaaggcct taattaggct
     6601 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     6661 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     6721 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     6781 gaagatcgga attcgccctt aagctagcta gttattaata gtaatcaatt acggggtcat
     6841 tagttcatag cccatatatg gagttccg
//
