LOCUS       sequence-442143-        7157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CMV driven inverted SaCas9. SaCas9 is floxed to render the system 
            cre-dependent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  AUTHORS   Kumar N, Stanford W, de Solis C, Aradhana, Abraham ND, Dao TJ, 
            Thaseen S, Sairavi A, Gonzalez CU, Ploski JE
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front Mol Neurosci. 2018 Nov 13;11:413. doi: 
            10.3389/fnmol.2018.00413. eCollection 2018.
  PUBMED    30483052
REFERENCE   2  (bases 1 to 7157)
  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: "Front Mol 
            Neurosci."; date: "2018-11-13"; volume: "11:413. doi"; pages: " 
            10.3389/fnmol.2018.00413. eCollection 2018"
COMMENT     Sequence Label: pJEP313-pAAV-CMV-SaCas9-DIO-pA
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     190..209
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   371..511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   382..511
                     /label=AAV2 ITR
     primer_bind     550..569
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(622..655)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(706..739)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      761..770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             773..793
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             818..3973
                     /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             3974..4021
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    4072..4105
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4156..4189
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(4249..4273)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4270..4473)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4299..4319)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4474..4777
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4801..4930
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4992..5580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5072..5091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5751..6542,6543..6611))
                     /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     6374..6393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6612..6716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6784..6802
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6840..6862)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6962..6981
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(6998..7157,1..296))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7135..7156)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
       61 actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa
      121 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
      181 gagaaaggaa gggaagaaag cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt
      241 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta
      301 tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      361 atcaggcgcc cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
      421 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
      481 ggagtggcca actccatcac taggggttcc tgcggccgct cggtccagtg aaaaaaatgc
      541 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
      601 ctcgagacta gtatcgaatt cataacttcg tatagcatac attatacgaa gttattcttt
      661 gcaccattct aaagaataac agtgataatt tctgggttaa ggcaaataac ttcgtatagg
      721 atactttata cgaagttatg ctagcgccac catgaccggt gccaccatgg ccccaaagaa
      781 gaagcggaag gtcggtatcc acggagtccc agcagccaag cggaactaca tcctgggcct
      841 ggacatcggc atcaccagcg tgggctacgg catcatcgac tacgagacac gggacgtgat
      901 cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa aacaacgagg gcaggcggag
      961 caagagaggc gccagaaggc tgaagcggcg gaggcggcat agaatccaga gagtgaagaa
     1021 gctgctgttc gactacaacc tgctgaccga ccacagcgag ctgagcggca tcaaccccta
     1081 cgaggccaga gtgaagggcc tgagccagaa gctgagcgag gaagagttct ctgccgccct
     1141 gctgcacctg gccaagagaa gaggcgtgca caacgtgaac gaggtggaag aggacaccgg
     1201 caacgagctg tccaccaaag agcagatcag ccggaacagc aaggccctgg aagagaaata
     1261 cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa
     1321 cagattcaag accagcgact acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc
     1381 ctaccaccag ctggaccaga gcttcatcga cacctacatc gacctgctgg aaacccggcg
     1441 gacctactat gagggacctg gcgagggcag ccccttcggc tggaaggaca tcaaagaatg
     1501 gtacgagatg ctgatgggcc actgcaccta cttccccgag gaactgcgga gcgtgaagta
     1561 cgcctacaac gccgacctgt acaacgccct gaacgacctg aacaatctcg tgatcaccag
     1621 ggacgagaac gagaagctgg aatattacga gaagttccag atcatcgaga acgtgttcaa
     1681 gcagaagaag aagcccaccc tgaagcagat cgccaaagaa atcctcgtga acgaagagga
     1741 tattaagggc tacagagtga ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta
     1801 ccacgacatc aaggacatta ccgcccggaa agagattatt gagaacgccg agctgctgga
     1861 tcagattgcc aagatcctga ccatctacca gagcagcgag gacatccagg aagaactgac
     1921 caatctgaac tccgagctga cccaggaaga gatcgagcag atctctaatc tgaagggcta
     1981 taccggcacc cacaacctga gcctgaaggc catcaacctg atcctggacg agctgtggca
     2041 caccaacgac aaccagatcg ctatcttcaa ccggctgaag ctggtgccca agaaggtgga
     2101 cctgtcccag cagaaagaga tccccaccac cctggtggac gacttcatcc tgagccccgt
     2161 cgtgaagaga agcttcatcc agagcatcaa agtgatcaac gccatcatca agaagtacgg
     2221 cctgcccaac gacatcatta tcgagctggc ccgcgagaag aactccaagg acgcccagaa
     2281 aatgatcaac gagatgcaga agcggaaccg gcagaccaac gagcggatcg aggaaatcat
     2341 ccggaccacc ggcaaagaga acgccaagta cctgatcgag aagatcaagc tgcacgacat
     2401 gcaggaaggc aagtgcctgt acagcctgga agccatccct ctggaagatc tgctgaacaa
     2461 ccccttcaac tatgaggtgg accacatcat ccccagaagc gtgtccttcg acaacagctt
     2521 caacaacaag gtgctcgtga agcaggaaga aaacagcaag aagggcaacc ggaccccatt
     2581 ccagtacctg agcagcagcg acagcaagat cagctacgaa accttcaaga agcacatcct
     2641 gaatctggcc aagggcaagg gcagaatcag caagaccaag aaagagtatc tgctggaaga
     2701 acgggacatc aacaggttct ccgtgcagaa agacttcatc aaccggaacc tggtggatac
     2761 cagatacgcc accagaggcc tgatgaacct gctgcggagc tacttcagag tgaacaacct
     2821 ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa
     2881 gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc gaggacgccc tgatcattgc
     2941 caacgccgat ttcatcttca aagagtggaa gaaactggac aaggccaaaa aagtgatgga
     3001 aaaccagatg ttcgaggaaa agcaggccga gagcatgccc gagatcgaaa ccgagcagga
     3061 gtacaaagag atcttcatca ccccccacca gatcaagcac attaaggact tcaaggacta
     3121 caagtacagc caccgggtgg acaagaagcc taatagagag ctgattaacg acaccctgta
     3181 ctccacccgg aaggacgaca agggcaacac cctgatcgtg aacaatctga acggcctgta
     3241 cgacaaggac aatgacaagc tgaaaaagct gatcaacaag agccccgaaa agctgctgat
     3301 gtaccaccac gacccccaga cctaccagaa actgaagctg attatggaac agtacggcga
     3361 cgagaagaat cccctgtaca agtactacga ggaaaccggg aactacctga ccaagtactc
     3421 caaaaaggac aacggccccg tgatcaagaa gattaagtat tacggcaaca aactgaacgc
     3481 ccatctggac atcaccgacg actaccccaa cagcagaaac aaggtcgtga agctgtccct
     3541 gaagccctac agattcgacg tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa
     3601 gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg aatagcaagt gctatgagga
     3661 agctaagaag ctgaagaaga tcagcaacca ggccgagttt atcgcctcct tctacaacaa
     3721 cgatctgatc aagatcaacg gcgagctgta tagagtgatc ggcgtgaaca acgacctgct
     3781 gaaccggatc gaagtgaaca tgatcgacat cacctaccgc gagtacctgg aaaacatgaa
     3841 cgacaagagg ccccccagga tcattaagac aatcgcctcc aagacccaga gcattaagaa
     3901 gtacagcaca gacattctgg gcaacctgta tgaagtgaaa tctaagaagc accctcagat
     3961 catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     4021 gggatccgat atctagatag ctggcctagg aattcacaag taaggcgcgc cataacttcg
     4081 tataatgtat gctatacgaa gttattaaga ggtttcatat tgctaatagc agctacaatc
     4141 cagctaccat tctgcataac ttcgtataaa gtatcctata cgaagttatt ccggagtcga
     4201 ccggtgtctt ctatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt
     4261 cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     4321 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     4381 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     4441 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     4501 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     4561 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     4621 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     4681 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     4741 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccacgc gtgcggccgc
     4801 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4861 ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4921 tgcctgcagg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4981 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5041 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5101 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5161 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5221 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5281 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5341 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5401 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5461 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5521 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5581 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5641 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5701 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6601 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6661 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6721 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     6781 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     6841 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     6901 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     6961 gcggcatcag agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg
     7021 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc
     7081 ggcaaaatcc cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt
     7141 tggaacaaga gtccact
//
