LOCUS       sequence-337255-        6867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector carrying 2 copies of a guide RNA targeting the mouse 
            PCSK9 mRNA, expressed from human and mouse U6 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6867)
  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 6867)
  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 (mPCSK9)
FEATURES             Location/Qualifiers
     source          1..6867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(580..893)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(641..661)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        complement(1299..1547)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1357..1376)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1527..1547)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    1549..1756
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1745..1762
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             1829..1840
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2238..2258
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2328..3038)
                     /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(2606..2625)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2875..2893
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3035..3044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3065..3084)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          3067..3199
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(3252..3455)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3281..3301)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3456..3759
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   3900..4029
                     /label=AAV2 ITR
     primer_bind     complement(4067..4083)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4067..4083)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4080..4102)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4091..4107
                     /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(4115..4121,4122..4139,4140..4145))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4160..4181
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4469..5057)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4549..4568)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5228..6019,6020..6088))
                     /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     5851..5870
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6089..6193)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6219..6674)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6356..6377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6568..6587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6801..6823
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6815..6832
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6816..6832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   join(6851..6867,1..113)
                     /label=AAV2 ITR
ORIGIN
        1 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
       61 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt
      121 aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga tccttatcgg
      181 gaattcgccc ttaactaaaa agcgtggact gtaccctcca cttttatccg ggcttgggac
      241 cggcattggc agtgttctac agtccacgcc gactgatggc agtttttttt ttggtgtctc
      301 accctgggta cccattcctg gtgtatggaa gcacctcctt cacggtcagg gggcctgtgc
      361 ttggctttct gccatcagac attaagctgt ggctggctct ggccagctgc tccagtgtac
      421 cagaacctga ggatgctcgc tgcaaggcct cagttctcag gccttagggt gtatttgtct
      481 ttcaggaagt tttttttttc tgccatcagt cggcatggtt aggcggtttc ccgccccctc
      541 ccattttatc cgggcttggg accggcgact gatggcccaa acaaggcttt tctccaaggg
      601 atatttatag tctcaaaaca cacaattact ttacagttag ggtgagtttc cttttgtgct
      661 gttttttaaa ataataattt agtatttgta tctcttatag aaatccaagc ctatcatgta
      721 aaatgtagct agtattaaaa agaacagatt atctgtcttt tatcgcacat taagcctcta
      781 tagttactag gaaatattat atgcaaatta accggggcag gggagtagcc gagcttctcc
      841 cacaagtctg tgcgaggggg ccggcgcggg cctagagatg gcggcgtcgg atctatctga
      901 gactgggcga ctcagtacga aaaaagcgtg gactgtaccc tccactttta tccgggcttg
      961 ggaccggcat tggcagtgtt ctacagtcca cgccgactga tggcagtttt tttttggtgt
     1021 ctcaccctgg gtacccattc ctggtgtatg gaagcacctc cttcacggtc agggggcctg
     1081 tgcttggctt tctgccatca gacattaagc tgtggctggc tctggccagc tgctccagtg
     1141 taccagaacc tgaggatgct cgctgcaagg cctcagttct caggccttag ggtgtatttg
     1201 tctttcagga agtttttttt tctgccatca gtcggcatgg ttaggcggtt tcccgccccc
     1261 tcccatttta tccgggcttg ggaccggcga ctgatggcgg tgtttcgtcc tttccacaag
     1321 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
     1381 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
     1441 ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc
     1501 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcgtc cccagcatgc
     1561 ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata
     1621 gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg
     1681 ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg
     1741 caactagaag gcacagtcga ggcagatctc gaagacgcgg aagaggccgc agagccggca
     1801 gcaggccgcg ggaaggaagg tccgctggat tgagggccga agggacgtag cagaaggacg
     1861 tcccgcgcag aatccaggtg gcaacacagg cgagcagcca aggaaaggac gatgatttcc
     1921 ccgacaacac cacggaattg tcagtgccca acagccgagc ccctgtccag cagcgggcaa
     1981 ggcaggcggc gatgagttcc gccgtggcaa tagggagggg gaaagcgaaa gtcccggaaa
     2041 ggagctgaca ggtggtggca atgccccaac cagtgggggt tgcgtcagca aacacagtgc
     2101 acaccacgcc acgttgcctg acaacgggcc acaactcctc ataaagagac agcaaccagg
     2161 atttatacaa ggaggagaaa atgaaagcca tacgggaagc aatagcatga tacaaaggca
     2221 ttaaagcagc gtatccacat agcgtaaaag gagcaacata gttaagaata ccagtcaatc
     2281 tttcacaaat tttgtaatcc agaggttgat tggatccaag ctccggttta cttgtacagc
     2341 tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg
     2401 tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc tccgggcagc
     2461 tgcacgggct tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc
     2521 ttcagcttca gcctctgctt gatctcgccc ttcagagcgc cgtcctcggg gtacatccgc
     2581 tcggaggagg cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag
     2641 ttggtgccgc gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg
     2701 gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg
     2761 gggaaggaca gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag
     2821 ccgtacatga actgagggga caggatgtcc caggcgaagg gcagggggcc acccttggtc
     2881 accttcagct tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc
     2941 tcgaactcgt ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg
     3001 atgatggcca tgttatcctc ctcgcccttg ctcaccatgg tggcggcgct agggccgcct
     3061 ggacacctgt ggagagaaag gcaaagtgga tgtcagtaag accaataggt gcctatcaga
     3121 aacgcaagag tcttctctgt ctcgacaagc ccagtttcta ttggtctcct taaacctgtc
     3181 ttgtaacctt gatacttacc tgcccagtgc ctcacgacca acttctgcag gatctgacgg
     3241 ttcactaaac cagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
     3301 gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
     3361 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
     3421 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg
     3481 tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg
     3541 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     3601 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
     3661 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     3721 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     3781 tgaactaatg accccgtaat tgattactat taataactag ctagcttaag ggcgaattcc
     3841 gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa
     3901 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     3961 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4021 agcgcgcagc ctaattaagg ccttaattaa atctggcgta atcatggtca tagctgtttc
     4081 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4141 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4201 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4261 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4321 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4381 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4441 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4501 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4561 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4621 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4681 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4741 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4801 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4861 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4921 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4981 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5041 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5101 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5161 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5221 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5281 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5341 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5401 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5461 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5521 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5581 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5641 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5701 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5761 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5821 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5881 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5941 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6001 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6061 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6121 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6181 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6241 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6301 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6361 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6421 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6481 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6541 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6601 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6661 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     6721 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     6781 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattagg
     6841 ttaattaagg ctgcgcgctc gctcgct
//
