LOCUS       sequence-214985-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 gRNA expression cassette followed by 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 7522)
  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 7522)
  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: pJEP314-pAAV-U6SaCas9gRNA(SapI)-CMV-SaCas9-DIO-pA
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        423..498
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        519..822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        823..1026
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     977..997
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1023..1047
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(1107..1140)
                     /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)."
     protein_bind    complement(1191..1224)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1275..1322)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1323..4478)
                     /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             complement(4503..4523)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4526..4535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4557..4590
                     /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)."
     protein_bind    4641..4674
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4727..4746)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4785..4925
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4785..4914
                     /label=AAV2 ITR
     rep_origin      5000..5455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5087..5106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5297..5318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5472..5491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5591..5613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5651..5669)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5737..5841
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5842..5910,5911..6702)
                     /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     complement(6060..6079)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6873..7461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7362..7381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggaaga gcgagctctt
      421 ctgttttagt actctggaaa cagaatctac taaaacaagg caaaatgccg tgtttatctc
      481 gtcaacttgt tggcgagatt tttacgcgtg gagttccgcg ttacataact tacggtaaat
      541 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      601 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      661 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      721 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      781 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      841 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      901 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      961 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1021 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
     1081 catagaagac accggtcgac tccggaataa cttcgtatag gatactttat acgaagttat
     1141 gcagaatggt agctggattg tagctgctat tagcaatatg aaacctctta ataacttcgt
     1201 atagcataca ttatacgaag ttatggcgcg ccttacttgt gaattcctag gccagctatc
     1261 tagatatcgg atcccttttt cttttttgcc tggccggcct ttttcgtggc cgccggcctt
     1321 ttgccctttt tgatgatctg agggtgcttc ttagatttca cttcatacag gttgcccaga
     1381 atgtctgtgc tgtacttctt aatgctctgg gtcttggagg cgattgtctt aatgatcctg
     1441 gggggcctct tgtcgttcat gttttccagg tactcgcggt aggtgatgtc gatcatgttc
     1501 acttcgatcc ggttcagcag gtcgttgttc acgccgatca ctctatacag ctcgccgttg
     1561 atcttgatca gatcgttgtt gtagaaggag gcgataaact cggcctggtt gctgatcttc
     1621 ttcagcttct tagcttcctc atagcacttg ctattcactt cgtagtagtt ttcttttttg
     1681 atcacatcca gattcttcac ggtcacgaac ttgtacacgc cattgtccag gtacacgtcg
     1741 aatctgtagg gcttcaggga cagcttcacg accttgtttc tgctgttggg gtagtcgtcg
     1801 gtgatgtcca gatgggcgtt cagtttgttg ccgtaatact taatcttctt gatcacgggg
     1861 ccgttgtcct ttttggagta cttggtcagg tagttcccgg tttcctcgta gtacttgtac
     1921 aggggattct tctcgtcgcc gtactgttcc ataatcagct tcagtttctg gtaggtctgg
     1981 gggtcgtggt ggtacatcag cagcttttcg gggctcttgt tgatcagctt tttcagcttg
     2041 tcattgtcct tgtcgtacag gccgttcaga ttgttcacga tcagggtgtt gcccttgtcg
     2101 tccttccggg tggagtacag ggtgtcgtta atcagctctc tattaggctt cttgtccacc
     2161 cggtggctgt acttgtagtc cttgaagtcc ttaatgtgct tgatctggtg gggggtgatg
     2221 aagatctctt tgtactcctg ctcggtttcg atctcgggca tgctctcggc ctgcttttcc
     2281 tcgaacatct ggttttccat cacttttttg gccttgtcca gtttcttcca ctctttgaag
     2341 atgaaatcgg cgttggcaat gatcagggcg tcctcggcgt ggtgcttgta ccccttgttc
     2401 cgctctttct taaacttcca cttccgccgc agaaagctgg tgaagccgcc attgatggac
     2461 ttcactttca cgtccaggtt gttcactctg aagtagctcc gcagcaggtt catcaggcct
     2521 ctggtggcgt atctggtatc caccaggttc cggttgatga agtctttctg cacggagaac
     2581 ctgttgatgt cccgttcttc cagcagatac tctttcttgg tcttgctgat tctgcccttg
     2641 cccttggcca gattcaggat gtgcttcttg aaggtttcgt agctgatctt gctgtcgctg
     2701 ctgctcaggt actggaatgg ggtccggttg cccttcttgc tgttttcttc ctgcttcacg
     2761 agcaccttgt tgttgaagct gttgtcgaag gacacgcttc tggggatgat gtggtccacc
     2821 tcatagttga aggggttgtt cagcagatct tccagaggga tggcttccag gctgtacagg
     2881 cacttgcctt cctgcatgtc gtgcagcttg atcttctcga tcaggtactt ggcgttctct
     2941 ttgccggtgg tccggatgat ttcctcgatc cgctcgttgg tctgccggtt ccgcttctgc
     3001 atctcgttga tcattttctg ggcgtccttg gagttcttct cgcgggccag ctcgataatg
     3061 atgtcgttgg gcaggccgta cttcttgatg atggcgttga tcactttgat gctctggatg
     3121 aagcttctct tcacgacggg gctcaggatg aagtcgtcca ccagggtggt ggggatctct
     3181 ttctgctggg acaggtccac cttcttgggc accagcttca gccggttgaa gatagcgatc
     3241 tggttgtcgt tggtgtgcca cagctcgtcc aggatcaggt tgatggcctt caggctcagg
     3301 ttgtgggtgc cggtatagcc cttcagatta gagatctgct cgatctcttc ctgggtcagc
     3361 tcggagttca gattggtcag ttcttcctgg atgtcctcgc tgctctggta gatggtcagg
     3421 atcttggcaa tctgatccag cagctcggcg ttctcaataa tctctttccg ggcggtaatg
     3481 tccttgatgt cgtggtacac cttcaggttg gtgaactcgg gcttgccggt gctggtcact
     3541 ctgtagccct taatatcctc ttcgttcacg aggatttctt tggcgatctg cttcagggtg
     3601 ggcttcttct tctgcttgaa cacgttctcg atgatctgga acttctcgta atattccagc
     3661 ttctcgttct cgtccctggt gatcacgaga ttgttcaggt cgttcagggc gttgtacagg
     3721 tcggcgttgt aggcgtactt cacgctccgc agttcctcgg ggaagtaggt gcagtggccc
     3781 atcagcatct cgtaccattc tttgatgtcc ttccagccga aggggctgcc ctcgccaggt
     3841 ccctcatagt aggtccgccg ggtttccagc aggtcgatgt aggtgtcgat gaagctctgg
     3901 tccagctggt ggtaggcctt ctgcaccttc agcagctgtt tggcttcttt cacgtagtcg
     3961 ctggtcttga atctgttgat gctgccccgc acttcgccgt ctttcttcag ccgttccagc
     4021 tgcagttcgg ccacgtattt ctcttccagg gccttgctgt tccggctgat ctgctctttg
     4081 gtggacagct cgttgccggt gtcctcttcc acctcgttca cgttgtgcac gcctcttctc
     4141 ttggccaggt gcagcagggc ggcagagaac tcttcctcgc tcagcttctg gctcaggccc
     4201 ttcactctgg cctcgtaggg gttgatgccg ctcagctcgc tgtggtcggt cagcaggttg
     4261 tagtcgaaca gcagcttctt cactctctgg attctatgcc gcctccgccg cttcagcctt
     4321 ctggcgcctc tcttgctccg cctgccctcg ttgttttcca cgttggcctc tttgaacagc
     4381 cgcacgccgg catcgatcac gtcccgtgtc tcgtagtcga tgatgccgta gcccacgctg
     4441 gtgatgccga tgtccaggcc caggatgtag ttccgcttgg ctgctgggac tccgtggata
     4501 ccgaccttcc gcttcttctt tggggccatg gtggcaccgg tcatggtggc gctagcataa
     4561 cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat
     4621 tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatgaattc
     4681 gatactagtc tcgagtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4741 aatttcacaa ataaagcatt tttttcactg gaccgagcgg ccgcaggaac ccctagtgat
     4801 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4861 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4921 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4981 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5041 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5101 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5161 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     5221 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5281 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5341 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5401 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     5461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5521 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5581 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5641 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5701 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5761 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5821 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5881 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5941 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6001 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6061 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6121 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6181 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6241 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6301 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6361 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6421 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6481 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6541 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6601 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6661 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6721 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6781 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6841 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6901 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6961 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     7021 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7081 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7141 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7201 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7261 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7321 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7381 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7441 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7501 tgctggcctt ttgctcacat gt
//
