LOCUS       sequence-363023-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses saCas9 protein specifically in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  AUTHORS   Endo F, Kasai A, Soto JS, Yu X, Qu Z, Hashimoto H, Gradinaru V, 
            Kawaguchi R, Khakh BS
  TITLE     Molecular basis of astrocyte diversity and morphology across the CNS
            in health and disease.
  JOURNAL   Science. 2022 Nov 4;378(6619):eadc9020. doi: 
            10.1126/science.adc9020. Epub 2022 Nov 4.
  PUBMED    36378959
REFERENCE   2  (bases 1 to 7200)
  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: "Science."; 
            date: "2022-11-4"; volume: "378(6619):eadc9020. doi"; pages: " 
            10.1126/science.adc9020. Epub 2022 Nov 4"
COMMENT     Sequence Label: PZac2.1 gfaABC1D-NLS-SaCas9-3xHA-NLS bGH
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      836..845
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             848..868
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             893..4048
                     /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             4049..4096
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4103..4183
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4211..4228)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4217..4424
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4463..4603
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4463..4592
                     /label=AAV2 ITR
     rep_origin      4678..5133
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4765..4784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4975..4996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5150..5169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5269..5291
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5329..5347)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5415..5519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5520..5588,5589..6380)
                     /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"
     rep_origin      6551..7139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7040..7059
                     /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 gactcgagaa catatcctgg tgtggagtag gggacgctgc
      181 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      241 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      301 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      361 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      421 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      481 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      541 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      601 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      661 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
      721 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
      781 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca ccggtgccac
      841 catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa
      901 ctacatcctg ggcctggaca tcggcatcac cagcgtgggc tacggcatca tcgactacga
      961 gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa
     1021 cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc ggcatagaat
     1081 ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca gcgagctgag
     1141 cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga gcgaggaaga
     1201 gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg tgaacgaggt
     1261 ggaagaggac accggcaacg agctgtccac caaagagcag atcagccgga acagcaaggc
     1321 cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag acggcgaagt
     1381 gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca aacagctgct
     1441 gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct acatcgacct
     1501 gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct tcggctggaa
     1561 ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc ccgaggaact
     1621 gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg acctgaacaa
     1681 tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt tccagatcat
     1741 cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca aagaaatcct
     1801 cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc ccgagttcac
     1861 caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga ttattgagaa
     1921 cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca gcgaggacat
     1981 ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg agcagatctc
     2041 taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca acctgatcct
     2101 ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc tgaagctggt
     2161 gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg tggacgactt
     2221 catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga tcaacgccat
     2281 catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg agaagaactc
     2341 caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg
     2401 gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga tcgagaagat
     2461 caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca tccctctgga
     2521 agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca gaagcgtgtc
     2581 cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg
     2641 caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct acgaaacctt
     2701 caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga ccaagaaaga
     2761 gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact tcatcaaccg
     2821 gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc ggagctactt
     2881 cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca ccagctttct
     2941 gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc acgccgagga
     3001 cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac tggacaaggc
     3061 caaaaaagtg atggaaaacc agatgttcga ggaaaagcag gccgagagca tgcccgagat
     3121 cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca agcacattaa
     3181 ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata gagagctgat
     3241 taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga tcgtgaacaa
     3301 tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca acaagagccc
     3361 cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga agctgattat
     3421 ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa ccgggaacta
     3481 cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta agtattacgg
     3541 caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca gaaacaaggt
     3601 cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa
     3661 gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg aagtgaatag
     3721 caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg agtttatcgc
     3781 ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag tgatcggcgt
     3841 gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct accgcgagta
     3901 cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg cctccaagac
     3961 ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag tgaaatctaa
     4021 gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa aggccggcca
     4081 ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgctt acccatacga
     4141 tgttccagat tacgcttacc catacgatgt tccagattac gcttaagaat tcctagagct
     4201 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4261 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4321 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4381 agcaaggggg aggattggga agagaatagc aggcatgctg gggaggtacg aaacaccgga
     4441 gaccacggca ggtctcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4501 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4561 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4621 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4681 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4741 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4801 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4861 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4921 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4981 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     5041 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5101 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     5161 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5221 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5281 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5341 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5401 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5461 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5521 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5581 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5641 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5701 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5761 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5821 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5881 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5941 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6001 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6061 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6121 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6181 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     6241 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6301 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6361 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6421 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6481 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6541 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6601 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6661 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6721 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6781 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6841 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6901 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6961 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7021 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7081 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7141 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
