LOCUS       sequence-375580-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector for encoding and AAV packaging a human codon-optimized 
            High-fidelity RfxCas13d (hfCas13d) driven by EFS promoter, 
            Pcsk9-target guide RNAs compatible with RfxCas13d driven by hU6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  AUTHORS   Tong H, Huang J, Xiao Q, He B, Dong X, Liu Y, Yang X, Han D, Wang Z,
            Wang X, Ying W, Zhang R, Wei Y, Xu C, Zhou Y, Li Y, Cai M, Wang Q, 
            Xue M, Li G, Fang K, Zhang H, Yang H
  TITLE     High-fidelity Cas13 variants for targeted RNA degradation with 
            minimal collateral effects.
  JOURNAL   Nat Biotechnol. 2022 Aug 11. pii: 10.1038/s41587-022-01419-7. doi: 
            10.1038/s41587-022-01419-7.
  PUBMED    35953673
REFERENCE   2  (bases 1 to 6849)
  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 Aug 11. pii: 10.1038/s41587-022-01419-7. doi: 
            10.1038/s41587-022-01419-7."
COMMENT     Sequence Label: 
            AAV2-ITR-pEFS-NLS_hfCas13d(RfxCas13d_N2V8)_FLAG_NLS-pA-U6-DR-Pcsk9-s
            g5-DR-Pcsk9-sg6-DR-AAV2-ITR
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        186..397
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             414..434
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3342..3365
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3366..3386
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3387..3611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3639..3887
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3639..3659
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3810..3829
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3889..3924
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        3955..3990
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        4021..4056
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   4112..4252
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4112..4241
                     /label=AAV2 ITR
     rep_origin      4327..4782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4414..4433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4624..4645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4799..4818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4918..4940
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4978..4996)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5064..5168
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5169..5237,5238..6029)
                     /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(5387..5406)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6200..6788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6689..6708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gataggtctt gaaaggagtg ggaattggct ccggtgcccg
      181 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      241 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      301 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      361 cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggtgccacc atgcctaaaa
      421 agaaaagaaa ggtgggttct ggtatcgaga agaagaagag cttcgccaag ggcatgggag
      481 tgaagagcac cctggtgtcc ggctctaagg tgtacatgac cacatttgct gagggaagcg
      541 acgccaggct ggagaagatc gtggagggcg atagcatcag atccgtgaac gagggagagg
      601 ctttcagcgc cgagatggct gacaagaacg ctggctacaa gatcggaaac gccaagtttt
      661 cccacccaaa gggctacgcc gtggtggcta acaacccact gtacaccgga ccagtgcagc
      721 aggacatgct gggactgaag gagacactgg agaagaggta cttcggcgag tccgccgacg
      781 gaaacgataa catctgcatc caggtcatcc acaacatcct ggatatcgag aagatcctgg
      841 tggagtacat cacaaacgtg gtgtacgtgg tgaacaacat ctccggcctg gacaaggata
      901 tcatcggctt cggaaagttt tctaccgtgt acacatacga cgagttcaag gatccagagc
      961 accaccgggc cgcttttaac aacaacgaca agctgatcaa cgccatcaag gctcagtacg
     1021 acgagttcga taactttctg gataacccca ggctgggcta cttcggacag gctttctttt
     1081 ctaaggaggg cagaaactac atcatcaact acggaaacga gtgttacgac atcctggccc
     1141 tgctgagcgg actgaggcac tgggtggtgc acaacaacga ggaggagtct cggatcagcc
     1201 gcacctggct gtacaacctg gacaagaacc tggataacga gtacatctcc acactgaact
     1261 acctgtacga caggatcacc aacgagctga caaacagctt ctccaagaac tctgccgcta
     1321 acgtgaacta catcgctgag accctgggca tcaacccagc tgagttcgct gagcagtact
     1381 tcagattttc catcatgaag gagcagaaga acctgggctt caacatcaca aagctgagag
     1441 aagtgatgct ggacagaaag gatatgtccg agatcaggaa gaaccacaag gtgttcgatt
     1501 ctatcagaac caaggtgtac acaatgatgg actttgtgat ctacaggtac tacatcgagg
     1561 aggatgccaa ggtggccgct gccaacaaga gcctgcccga caacgagaag tctctgagcg
     1621 agaaggatat cttcgtgatc aacctgagag gctcctttaa cgacgatcag aaggacgctc
     1681 tgtactacga tgaggccaac aggatctgga gaaagctgga gaacatcatg cacaacatca
     1741 aggagttccg gggaaacaag acccgcgagt acaagaagaa ggacgctcca aggctgccta
     1801 ggatcctgcc tgctggaagg gacgtgagcg ccttcagcaa gctgatgtac gccctgacaa
     1861 tgtttctgga cggaaaggag atcaacgatc tgctgaccac actgatcaac aagttcgaca
     1921 acatccagtc ttttctgaaa gtgatgcctc tgatcggcgt gaacgctaag ttcgtggagg
     1981 agtacgcctt ctttaaggac agcgccaaga tcgctgatga gctgcggctg atcaagtcct
     2041 ttgccaggat gggagagcca atcgctgacg ctaggagagc tatgtacatc gatgccatcc
     2101 ggatcctggg aaccaacctg tcttacgacg agctgaaggc tctggccgac accttcagcc
     2161 tggatgagaa cggcaacaag ctgaagaagg gcaagcacgg aatgcgcaac ttcatcatca
     2221 acaacgtgat cagcaacaag cggtttcact acctgatcag atacggcgac ccagctcacc
     2281 tgcacgagat cgctaagaac gaggccgtgg tgaagttcgt gctgggacgg atcgccgata
     2341 tccagaagaa gcagggccag aacggaaaga accagatcga ccgctactac gagacctgca
     2401 tcggcaagga taagggaaag tccgtgtctg agaaggtgga cgctctgacc aagatcatca
     2461 caggcatgaa ctacgaccag ttcgataaga agagatctgt gatcgaggac accggaaggg
     2521 agaacgccga gagagagaag tttaagaaga tcatcagcct gtacctgaca gtgatctacc
     2581 acatcctgaa gaacatcgtg aacatcaacg ctagatacgt gatcggcttc cactgcgtgg
     2641 agcgcgatgc ccagctgtac aaggagaagg gatacgacat caacctgaag aagctggagg
     2701 agaagggctt tagctccgtg accaagctgt gcgctggaat cgacgagaca gcccccgaca
     2761 agaggaagga tgtggagaag gagatggccg agagagctaa ggagagcatc gactccctgg
     2821 agtctgctaa ccctaagctg tacgccaact acatcaagta ctccgatgag aagaaggccg
     2881 aggagttcac caggcagatc aacagagaga aggccaagac cgctctgaac gcctacctga
     2941 ggaacacaaa gtggaacgtg atcatccggg aggacctgct gcgcatcgat aacaagacct
     3001 gtacactgtt ccggaacaag gctgtgcacc tggaggtggc tcgctacgtg cacgcctaca
     3061 tcaacgacat cgccgaggtg aactcctact ttcagctgta ccactacatc atgcagagga
     3121 tcatcatgaa cgagagatac gagaagtcta gcggcaaggt gtctgagtac ttcgacgccg
     3181 tgaacgatga gaagaagtac aacgatagac tgctgaagct gctgtgcgtg cctttcggat
     3241 actgtatccc acggtttaag aacctgagca tcgaggccct gttcgaccgc aacgaggctg
     3301 ccaagtttga taaggagaag aagaaggtga gcggcaactc cgattacaaa gacgatgacg
     3361 ataagcccaa gaagaagagg aaagtcctgt gccttctagt tgccagccat ctgttgtttg
     3421 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3481 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3541 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3601 gggctctatg ggatgggctg gccttttgct cagctagcga gggcctattt cccatgattc
     3661 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     3721 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3781 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3841 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca agtaaacccc
     3901 taccaactgg tcggggtttg aaacgtgggt gccgtggctg tcacacttgc tcgccaagta
     3961 aacccctacc aactggtcgg ggtttgaaac cagcatcccg gccgctgacc acacctgcca
     4021 caagtaaacc cctaccaact ggtcggggtt tgaaactttt tttgaattct gatgcggtat
     4081 tttctcctta cgctgatgcg gtggcggccg caggaacccc tagtgatgga gttggccact
     4141 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4201 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4261 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4321 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4381 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4441 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4501 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4561 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4621 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4681 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4741 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4801 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4861 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4921 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4981 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5041 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5101 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5161 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5221 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5281 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5341 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5401 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5461 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5521 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5581 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5641 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5701 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5761 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5821 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5881 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5941 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6001 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6061 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6121 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6181 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6241 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6301 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6361 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6421 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6481 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6541 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6601 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6661 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6721 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6781 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6841 ctcacatgt
//
