LOCUS       sequence-345286-        7411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV EFS-HIVNES-dCas13bt1- (GGS)2-huADAR2(E488Q)-3xHA 
            BGHpolyA::U6-BpiI-Cas13bt1 DR (full REPAIR.t1 + crRNA expression for
            AAV production).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7411)
  AUTHORS   Kannan S, Altae-Tran H, Jin X, Madigan VJ, Oshiro R, Makarova KS, 
            Koonin EV, Zhang F
  TITLE     Compact RNA editors with small Cas13 proteins.
  JOURNAL   Nat Biotechnol. 2021 Aug 30. pii: 10.1038/s41587-021-01030-2. doi: 
            10.1038/s41587-021-01030-2.
  PUBMED    34462587
REFERENCE   2  (bases 1 to 7411)
  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. 2021 Aug 30. pii: 10.1038/s41587-021-01030-2. doi: 
            10.1038/s41587-021-01030-2."
COMMENT     Sequence Label: pAB2076 pAAV REPAIR.t1
FEATURES             Location/Qualifiers
     source          1..7411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      226..235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             247..273
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             3868..3948
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3955..3972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3961..4168
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(4232..4480)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4290..4309)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4460..4480)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4489..4629
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4489..4618
                     /label=AAV2 ITR
     rep_origin      4704..5159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4791..4810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5001..5022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5176..5195)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5295..5317
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5355..5373)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5441..5545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5546..5614,5615..6406)
                     /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(5764..5783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6577..7165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7066..7085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7227..7356
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggaaactta agcttgccac catgggatcc
      241 cttcaactgc ctccacttga aagactgaca ctgggatccg aattcgagaa catcaagaaa
      301 accagcaaca aagaggtgta cagcatcgag cagtacgagg gcgagaagaa gtggtgcttc
      361 gccatcgtgc tgaacagagc ccagaccaac ctggaagaga accccaagct gttcgagcag
      421 accctgacca gatttgagaa gatcatgaag caggactggt tcaacgagga aacgaagaag
      481 ctgatctacg agaaagagga agaaaacaaa gtcaaagagg aaatccagat cgccgccagc
      541 gagcggctga agaacctggc aaactacttc agcgcctacc tgcacgcccc tgactgcctg
      601 atcttcaacc ggaacgacac catccggatc atcatggaaa aggcctacga gaagtcccgc
      661 ttcgaggcca agaagaagca gcaagaggac atctccatcg agttccccga gctgtttgag
      721 gaagaggaca agatcacctc tgccggcgtg gtgttcttcg tcagcttttt tatcgagcgg
      781 cggttcctga accggctgat gggctatgtg cagggcttca gaaagacaga gggcgagtac
      841 aacatcacca gacaggtgtt cagcaagtac tgcctgaagg acagctacag cgtgcaggcc
      901 caggatcacg atgccgtgat gttcagagac atcctgggct acctgagcag agtgcccacc
      961 gagatctacc agcacatcaa gctgacccgg aagcggagcc aggatcagct gagcgagaga
     1021 aagaccgaca agttcatcct gttcgccctg aagtacctcg aggactacgg actgaaggac
     1081 ctggccgatt acaccgcctg ctttgccaga agcaagatca agagagagaa cgaggacacc
     1141 aaagagacag acggcaacaa gcacaagttc caccgcgaga agcccgtggt ggaaatccac
     1201 ttcgacaaag agaagcagga tcagttctac atcaagcgga acaacgtcat cctgaaggcc
     1261 cagaagaaag gcggccagag caacgtgttc cggatgggag tgtacgagct gaagtatctg
     1321 gtgctgctga gcctgctggg caaagccgaa gaggccatcc agagaatcga ccggtacatc
     1381 agcagcctga agaaacagct gccctacctg gataagatca gcaacgaaga gatccagaag
     1441 tccatcaact ttctgcccag attcgtgcgg agccggctgg gactgctgca agtggatgat
     1501 gagaagagac tgaaaacccg gctggaatac gtgaaggcca agtggaccga taagaaagag
     1561 ggcagcagaa agctggaact gcaccggaag ggcagagata tcctgcggta tatcaacgag
     1621 agatgcgaca gacccctgag ccggaaagag tataacaaca ttctgaagtt catcgtgaac
     1681 aaggacttcg ccggcttcta caacgagctg gaagaactga agcggaccag acggctggac
     1741 aagaacatca tccagaagct gagcggccac accacactga acgctctgca cgagcgagtg
     1801 tgtgacctgg tcctgcaaga gctgggctct ctgcagtccg agaacctgaa agagtacatc
     1861 ggactgatcc ccaaagaaga gaaagaagtc accttccgcg agaaagtgga cagaatcctg
     1921 gaacagcctg tggtgtacaa gggcttcctg agatacgagt tcttcaaaga agataagaaa
     1981 tccttcgcca ggctggtgga agaggctatc aagaccaagt ggagcgactt cgacatccct
     2041 ctgggcgaag agtactacaa catccctagc ctggacagat tcgaccggac caacaagaag
     2101 ctgtacgaga cactggccat ggaccggctg tgtctgatga tggccagaca gtactacctg
     2161 cggctgaacg agaagctggc cgagaaggca cagcacatct actggaaaaa agaggacggc
     2221 cgggaagtca tcatcttcaa gttccagaat ccgaaagagc agaagaagtc cttcagcatc
     2281 aggttcagca tcctggacta caccaagatg tacgtgatgg acgaccccga gttcctgagc
     2341 aggctgtggg agtacttcat ccctaaagag gccaaagaga tcgactacca caagcactac
     2401 gccagagcct tcgacaagta caccaacctg cagaaagaag gcatcgacgc catcctgaaa
     2461 ctggaaggca gaatcatcga gcgccggaag atcaagcccg ccaagaacta catcgagttt
     2521 caagagatta tgaatcggag cggctacaac aacgaccagc aggttgccct gaagagagtg
     2581 gcgaacgccc tgctggccta caacctgaac ttcgagagag agcacctgaa gcggttctac
     2641 ggcgtcgtga agagagaggg catcgagaaa aaatggtccc tgatcgtggg agggtcaggc
     2701 ggtagtcagc tgcatttacc gcaggtttta gctgacgctg tctcacgcct ggtcctgggt
     2761 aagtttggtg acctgaccga caacttctcc tcccctcacg ctcgcagaaa agtgctggct
     2821 ggagtcgtca tgacaacagg cacagatgtt aaagatgcca aggtgataag tgtttctaca
     2881 ggaacaaaat gtattaatgg tgaatacatg agtgatcgtg gccttgcatt aaatgactgc
     2941 catgcagaaa taatatctcg gagatccttg ctcagatttc tttatacaca acttgagctt
     3001 tacttaaata acaaagatga tcaaaaaaga tccatctttc agaaatcaga gcgagggggg
     3061 tttaggctga aggagaatgt ccagtttcat ctgtacatca gcacctctcc ctgtggagat
     3121 gccagaatct tctcaccaca tgagccaatc ctggaagaac cagcagatag acacccaaat
     3181 cgtaaagcaa gaggacagct acggaccaaa atagagtctg gtcaggggac gattccagtg
     3241 cgctccaatg cgagcatcca aacgtgggac ggggtgctgc aaggggagcg gctgctcacc
     3301 atgtcctgca gtgacaagat tgcacgctgg aacgtggtgg gcatccaggg atcactgctc
     3361 agcattttcg tggagcccat ttacttctcg agcatcatcc tgggcagcct ttaccacggg
     3421 gaccaccttt ccagggccat gtaccagcgg atctccaaca tagaggacct gccacctctc
     3481 tacaccctca acaagccttt gctcagtggc atcagcaatg cagaagcacg gcagccaggg
     3541 aaggccccca acttcagtgt caactggacg gtaggcgact ccgctattga ggtcatcaac
     3601 gccacgactg ggaaggatga gctgggccgc gcgtcccgcc tgtgtaagca cgcgttgtac
     3661 tgtcgctgga tgcgtgtgca cggcaaggtt ccctcccact tactacgctc caagattacc
     3721 aagcccaacg tgtaccatga gtccaagctg gcggcaaagg agtaccaggc cgccaaggcg
     3781 cgtctgttca cagccttcat caaggcgggg ctgggggcct gggtggagaa gcccaccgag
     3841 caggaccagt tctcactcac gggatcctac ccatacgatg ttccagatta cgcttatccc
     3901 tacgacgtgc ctgattatgc atacccatat gatgtccccg actatgccta acgacctcga
     3961 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     4021 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     4081 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     4141 gggaagagaa tagcaggcat gctggggaaa aaaagctgta atcaccccac aaatcggagg
     4201 cttcttcagc tgagaccgtc ccagggtctc tggtgtttcg tcctttccac aagatatata
     4261 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
     4321 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
     4381 ctaatatctt tgtgtttaca gtcaaattaa ttccaattat ctctctaaca gccttgtatc
     4441 gtatatgcaa atatgaagga atcatgggaa ataggccctc gcggccgcag gaacccctag
     4501 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4561 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4621 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4681 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5041 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5101 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5161 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5221 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5281 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5341 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5401 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5761 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6061 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6241 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     6301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6721 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7201 cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga
     7261 ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     7321 gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagata ggtcttgaaa
     7381 ggagtgggaa ttggctccgg tgcccgtcag t
//
