LOCUS       sequence-459842-        6906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SCP1 promoter-driven SaCas9 with truncated CMV enhancer and short 
            synthetic terminator sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6906)
  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 6906)
  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.minCMVe-SCP1-SaCas9-W3SynpA
FEATURES             Location/Qualifiers
     source          1..6906
                     /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
     regulatory      1184..1193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1196..1216
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1241..4396
                     /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             4397..4444
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4451..4531
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4596..4616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4794..4842
                     /note="synthetic polyadenylation signal"
     repeat_region   4861..5001
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4861..4990
                     /label=AAV2 ITR
     rep_origin      complement(5063..5651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5143..5162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5822..6613,6614..6682))
                     /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     6445..6464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6683..6787)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6855..6873
                     /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 cctcgagcca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      901 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      961 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     1021 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     1081 catcgctatt accatggtac ttatataagg gggtgggggc gcgttcgtcc tcagtcgcga
     1141 tcgaacactc gagccgagca gacgtgccta cggaccgacc ggtgccacca tggccccaaa
     1201 gaagaagcgg aaggtcggta tccacggagt cccagcagcc aagcggaact acatcctggg
     1261 cctggacatc ggcatcacca gcgtgggcta cggcatcatc gactacgaga cacgggacgt
     1321 gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg
     1381 gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc agagagtgaa
     1441 gaagctgctg ttcgactaca acctgctgac cgaccacagc gagctgagcg gcatcaaccc
     1501 ctacgaggcc agagtgaagg gcctgagcca gaagctgagc gaggaagagt tctctgccgc
     1561 cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac
     1621 cggcaacgag ctgtccacca aagagcagat cagccggaac agcaaggccc tggaagagaa
     1681 atacgtggcc gaactgcagc tggaacggct gaagaaagac ggcgaagtgc ggggcagcat
     1741 caacagattc aagaccagcg actacgtgaa agaagccaaa cagctgctga aggtgcagaa
     1801 ggcctaccac cagctggacc agagcttcat cgacacctac atcgacctgc tggaaacccg
     1861 gcggacctac tatgagggac ctggcgaggg cagccccttc ggctggaagg acatcaaaga
     1921 atggtacgag atgctgatgg gccactgcac ctacttcccc gaggaactgc ggagcgtgaa
     1981 gtacgcctac aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac
     2041 cagggacgag aacgagaagc tggaatatta cgagaagttc cagatcatcg agaacgtgtt
     2101 caagcagaag aagaagccca ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga
     2161 ggatattaag ggctacagag tgaccagcac cggcaagccc gagttcacca acctgaaggt
     2221 gtaccacgac atcaaggaca ttaccgcccg gaaagagatt attgagaacg ccgagctgct
     2281 ggatcagatt gccaagatcc tgaccatcta ccagagcagc gaggacatcc aggaagaact
     2341 gaccaatctg aactccgagc tgacccagga agagatcgag cagatctcta atctgaaggg
     2401 ctataccggc acccacaacc tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg
     2461 gcacaccaac gacaaccaga tcgctatctt caaccggctg aagctggtgc ccaagaaggt
     2521 ggacctgtcc cagcagaaag agatccccac caccctggtg gacgacttca tcctgagccc
     2581 cgtcgtgaag agaagcttca tccagagcat caaagtgatc aacgccatca tcaagaagta
     2641 cggcctgccc aacgacatca ttatcgagct ggcccgcgag aagaactcca aggacgccca
     2701 gaaaatgatc aacgagatgc agaagcggaa ccggcagacc aacgagcgga tcgaggaaat
     2761 catccggacc accggcaaag agaacgccaa gtacctgatc gagaagatca agctgcacga
     2821 catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc cctctggaag atctgctgaa
     2881 caaccccttc aactatgagg tggaccacat catccccaga agcgtgtcct tcgacaacag
     2941 cttcaacaac aaggtgctcg tgaagcagga agaaaacagc aagaagggca accggacccc
     3001 attccagtac ctgagcagca gcgacagcaa gatcagctac gaaaccttca agaagcacat
     3061 cctgaatctg gccaagggca agggcagaat cagcaagacc aagaaagagt atctgctgga
     3121 agaacgggac atcaacaggt tctccgtgca gaaagacttc atcaaccgga acctggtgga
     3181 taccagatac gccaccagag gcctgatgaa cctgctgcgg agctacttca gagtgaacaa
     3241 cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc agctttctgc ggcggaagtg
     3301 gaagtttaag aaagagcgga acaaggggta caagcaccac gccgaggacg ccctgatcat
     3361 tgccaacgcc gatttcatct tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat
     3421 ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg cccgagatcg aaaccgagca
     3481 ggagtacaaa gagatcttca tcacccccca ccagatcaag cacattaagg acttcaagga
     3541 ctacaagtac agccaccggg tggacaagaa gcctaataga gagctgatta acgacaccct
     3601 gtactccacc cggaaggacg acaagggcaa caccctgatc gtgaacaatc tgaacggcct
     3661 gtacgacaag gacaatgaca agctgaaaaa gctgatcaac aagagccccg aaaagctgct
     3721 gatgtaccac cacgaccccc agacctacca gaaactgaag ctgattatgg aacagtacgg
     3781 cgacgagaag aatcccctgt acaagtacta cgaggaaacc gggaactacc tgaccaagta
     3841 ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag tattacggca acaaactgaa
     3901 cgcccatctg gacatcaccg acgactaccc caacagcaga aacaaggtcg tgaagctgtc
     3961 cctgaagccc tacagattcg acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt
     4021 gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca agtgctatga
     4081 ggaagctaag aagctgaaga agatcagcaa ccaggccgag tttatcgcct ccttctacaa
     4141 caacgatctg atcaagatca acggcgagct gtatagagtg atcggcgtga acaacgacct
     4201 gctgaaccgg atcgaagtga acatgatcga catcacctac cgcgagtacc tggaaaacat
     4261 gaacgacaag aggcccccca ggatcattaa gacaatcgcc tccaagaccc agagcattaa
     4321 gaagtacagc acagacattc tgggcaacct gtatgaagtg aaatctaaga agcaccctca
     4381 gatcatcaaa aagggcaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     4441 aaagggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg ttccagatta
     4501 cgcttaccca tacgatgttc cagattacgc ttaagaattc ataatcaacc tctggattac
     4561 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4621 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4681 tccttgtata aatcctggtt agttcttgcc acggcggaac tcatcgccgc ctgccttgcc
     4741 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt tataataaaa
     4801 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgcaaggtac cttgggccgc
     4861 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4921 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg agcggcctca gtgagcgagc
     4981 gagcgcgcag ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5041 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5101 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5161 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5221 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5281 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5341 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5401 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5461 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5521 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5581 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5641 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5701 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5761 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5821 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5881 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5941 ccagtgctgc aatgataccg cggcttccac gctcaccggc tccagattta tcagcaataa
     6001 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6061 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6121 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6181 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6241 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6301 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6361 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6421 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6481 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6541 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6601 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6661 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6721 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6781 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6841 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     6901 acggtg
//
