LOCUS       Exported                7513 bp ds-DNA     circular SYN 11-JUN-2025
DEFINITION  pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA Sequencing 
            Result.
ACCESSION   .
VERSION     .
KEYWORDS    M167-2 pGFAP(pr)X601-AAV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7513)
  AUTHORS   Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
            Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F.
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature 2015;520:186-91.
REFERENCE   2  (bases 1 to 7513)
  AUTHORS   Robbie
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jun 11, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Source: https://www.addgene.org/61591
            4 verification sequences were imported and aligned.
            ORIGDB|GenBank
FEATURES             Location/Qualifiers
     source          1..7513
                     /organism="pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::Bsa
                     I-sgRNA Sequencing Result"
                     /mol_type="other DNA"
                     /note="/vntifkey=98"
     misc_feature    40..149
                     /label=ITR
                     /note="/vntifkey=21"
     promoter        150..832
                     /label=GFAP
                     /note="/vntifkey=29"
     regulatory      834..843
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             846..866
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             891..4046
                     /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             4047..4094
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4101..4181
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4209..4226)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4215..4422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4429..4669
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4429..4449
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4600..4619
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4698..4773
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4787..4916
                     /label=AAV2 ITR
     repeat_region   4787..4916
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4991..5446
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5728..5832
                     /gene="bla"
                     /label=AmpR promoter
     gene            5833..6693
                     /gene="Ampicillin"
                     /label=Ampicillin
                     /note="/vntifkey=60"
     CDS             5833..6693
                     /codon_start=1
                     /label=Ampr
                     /note="/vntifkey=4"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6864..7452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgagga acatatcctg gtgtggagta ggggacgctg
      181 ctctgacaga ggctcggggg cctgagctgg ctctgtgagc tggggaggag gcagacagcc
      241 aggccttgtc tgcaagcaga cctggcagca ttgggctggc cgccccccag ggcctcctct
      301 tcatgcccag tgaatgactc accttggcac agacacaatg ttcggggtgg gcacagtgcc
      361 tgcttcccgc cgcaccccag cccccctcaa atgccttccg agaagcccat tgagcagggg
      421 gcttgcattg caccccagcc tgacagcctg gcatcttggg ataaaagcag cacagccccc
      481 taggggctgc ccttgctgtg tggcgccacc ggcggtggag aacaaggctc tattcagcct
      541 gtgcccagga aaggggatca ggggatgccc aggcatggac agtgggtggc agggggggag
      601 aggagggctg tctgcttccc agaagtccaa ggacacaaat gggtgagggg agagctctcc
      661 ccatagctgg gctgcggccc aaccccaccc cctcaggcta tgccaggggg tgttgccagg
      721 ggcacccggg catcgccagt ctagcccact ccttcataaa gccctcgcat cccaggagcg
      781 agcagagcca gagcaggttg gagaggagac gcatcacctc cgctgctcgc ggtgccacca
      841 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc aagcggaact
      901 acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc gactacgaga
      961 cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg
     1021 agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc
     1081 agagagtgaa gaagctgctg ttcgactaca acctgctgac cgaccacagc gagctgagcg
     1141 gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca gaagctgagc gaggaagagt
     1201 tctctgccgc cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg
     1261 aagaggacac cggcaacgag ctgtccacca aagagcagat cagccggaac agcaaggccc
     1321 tggaagagaa atacgtggcc gaactgcagc tggaacggct gaagaaagac ggcgaagtgc
     1381 ggggcagcat caacagattc aagaccagcg actacgtgaa agaagccaaa cagctgctga
     1441 aggtgcagaa ggcctaccac cagctggacc agagcttcat cgacacctac atcgacctgc
     1501 tggaaacccg gcggacctac tatgagggac ctggcgaggg cagccccttc ggctggaagg
     1561 acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc gaggaactgc
     1621 ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc
     1681 tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc cagatcatcg
     1741 agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa gaaatcctcg
     1801 tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc gagttcacca
     1861 acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt attgagaacg
     1921 ccgagctgct ggatcagatt gccaagatcc tgaccatcta ccagagcagc gaggacatcc
     1981 aggaagaact gaccaatctg aactccgagc tgacccagga agagatcgag cagatctcta
     2041 atctgaaggg ctataccggc acccacaacc tgagcctgaa ggccatcaac ctgatcctgg
     2101 acgagctgtg gcacaccaac gacaaccaga tcgctatctt caaccggctg aagctggtgc
     2161 ccaagaaggt ggacctgtcc cagcagaaag agatccccac caccctggtg gacgacttca
     2221 tcctgagccc cgtcgtgaag agaagcttca tccagagcat caaagtgatc aacgccatca
     2281 tcaagaagta cggcctgccc aacgacatca ttatcgagct ggcccgcgag aagaactcca
     2341 aggacgccca gaaaatgatc aacgagatgc agaagcggaa ccggcagacc aacgagcgga
     2401 tcgaggaaat catccggacc accggcaaag agaacgccaa gtacctgatc gagaagatca
     2461 agctgcacga catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc cctctggaag
     2521 atctgctgaa caaccccttc aactatgagg tggaccacat catccccaga agcgtgtcct
     2581 tcgacaacag cttcaacaac aaggtgctcg tgaagcagga agaaaacagc aagaagggca
     2641 accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac gaaaccttca
     2701 agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc aagaaagagt
     2761 atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc atcaaccgga
     2821 acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg agctacttca
     2881 gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc agctttctgc
     2941 ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac gccgaggacg
     3001 ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg gacaaggcca
     3061 aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg cccgagatcg
     3121 aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag cacattaagg
     3181 acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga gagctgatta
     3241 acgacaccct gtactccacc cggaaggacg acaagggcaa caccctgatc gtgaacaatc
     3301 tgaacggcct gtacgacaag gacaatgaca agctgaaaaa gctgatcaac aagagccccg
     3361 aaaagctgct gatgtaccac cacgaccccc agacctacca gaaactgaag ctgattatgg
     3421 aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc gggaactacc
     3481 tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag tattacggca
     3541 acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga aacaaggtcg
     3601 tgaagctgtc cctgaagccc tacagattcg acgtgtacct ggacaatggc gtgtacaagt
     3661 tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca
     3721 agtgctatga ggaagctaag aagctgaaga agatcagcaa ccaggccgag tttatcgcct
     3781 ccttctacaa caacgatctg atcaagatca acggcgagct gtatagagtg atcggcgtga
     3841 acaacgacct gctgaaccgg atcgaagtga acatgatcga catcacctac cgcgagtacc
     3901 tggaaaacat gaacgacaag aggcccccca ggatcattaa gacaatcgcc tccaagaccc
     3961 agagcattaa gaagtacagc acagacattc tgggcaacct gtatgaagtg aaatctaaga
     4021 agcaccctca gatcatcaaa aagggcaaaa ggccggcggc cacgaaaaag gccggccagg
     4081 caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg
     4141 ttccagatta cgcttaccca tacgatgttc cagattacgc ttaagaattc ctagagctcg
     4201 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4261 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4321 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4381 caagggggag gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt
     4441 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     4501 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     4561 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     4621 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga
     4681 gaccacggca ggtctcagtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa
     4741 tgccgtgttt atctcgtcaa cttgttggcg agatttttgc ggccgcagga acccctagtg
     4801 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4861 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagggcg
     4921 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4981 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5041 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     5101 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5161 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5221 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5281 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5341 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5401 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5461 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5521 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5581 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5641 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5701 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5761 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5821 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5881 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5941 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6001 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6061 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6121 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6181 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6241 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6301 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6361 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6421 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6481 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6541 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6601 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6661 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6721 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6781 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6841 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6901 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6961 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     7021 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7081 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7141 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7201 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7261 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7321 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7381 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7441 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7501 tttgctcaca tgt
//
