LOCUS       sequence-459832-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minBG-driven SaCas9, with short synthetic terminator sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  AUTHORS   Coughlin GM, Borsos M, Barcelona BH, Appling N, Mayfield AMH, Mackey
            ED, Eser RA, Jackson CR, Chen X, Kumar SR, Gradinaru V
  TITLE     Spatial genomics of AAV vectors reveals mechanism of transcriptional
            crosstalk that enables targeted delivery of large genetic cargo.
  JOURNAL   Nat Biotechnol. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4.
  PUBMED    40113953
REFERENCE   2  (bases 1 to 6770)
  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. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4."
COMMENT     Sequence Label: pAAV.minBG-CI-SaCas9-W3SynpA
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     intron          909..1041
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1048..1057
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1060..1080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1105..4260
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4261..4308
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4315..4395
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4460..4480)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4658..4706
                     /note="synthetic polyadenylation signal"
     repeat_region   4725..4865
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4725..4854
                     /label=AAV2 ITR
     rep_origin      complement(4927..5515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5007..5026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5686..6477,6478..6546))
                     /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     6309..6328
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6547..6651)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6719..6737
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cctcgagagc ccgggctggg cataaaagtc agggcagagc catctattgc ttacatttgc
      901 ttctagaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      961 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
     1021 actttgcctt tctctccaca gaccggtgcc accatggccc caaagaagaa gcggaaggtc
     1081 ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctgga catcggcatc
     1141 accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg
     1201 cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc
     1261 agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac
     1321 tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg
     1381 aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc
     1441 aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc
     1501 accaaagagc agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg
     1561 cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc
     1621 agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg
     1681 gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag
     1741 ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg
     1801 atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc
     1861 gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag
     1921 aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag
     1981 cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac
     2041 agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag
     2101 gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag
     2161 atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc
     2221 gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac
     2281 aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac
     2341 cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag
     2401 aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc
     2461 ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac
     2521 atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag
     2581 atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc
     2641 aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag
     2701 tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat
     2761 gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg
     2821 ctcgtgaagc aggaagaaaa cagcaagaag ggcaaccgga ccccattcca gtacctgagc
     2881 agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag
     2941 ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac
     3001 aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc
     3061 agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg
     3121 aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag
     3181 cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc
     3241 atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc
     3301 gaggaaaagc aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc
     3361 ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac
     3421 cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag
     3481 gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat
     3541 gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac
     3601 ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc
     3661 ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac
     3721 ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc
     3781 accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga
     3841 ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg
     3901 atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg
     3961 aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag
     4021 atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa
     4081 gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc
     4141 cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac
     4201 attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc
     4261 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atcctaccca
     4321 tacgatgttc cagattacgc ttacccatac gatgttccag attacgctta cccatacgat
     4381 gttccagatt acgcttaaga attcataatc aacctctgga ttacaaaatt tgtgaaagat
     4441 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     4501 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     4561 ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     4621 ctcggctgtt gggcactgac aattccgtgg tgtttataat aaaatatctt tattttcatt
     4681 acatctgtgt gttggttttt tgtgtgcaag gtaccttggg ccgcaggaac ccctagtgat
     4741 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4801 cgcccgacgc ccgggctttg cccgagcggc ctcagtgagc gagcgagcgc gcagctgcct
     4861 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4921 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4981 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5041 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5101 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5161 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5221 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5281 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5341 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5401 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5461 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5521 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5581 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5641 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5701 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5761 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5821 accgcggctt ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5881 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5941 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6001 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6061 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6121 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6181 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6241 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6301 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6361 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6421 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6481 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6541 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6601 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6661 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6721 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
//
