LOCUS       sequence-214986-        7524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 SaCas9 gRNA expression cassette containing a gRNA targeting the 
            CREB gene, 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 7524)
  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 7524)
  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: pJEP315-pAAV-U6SaCas9gRNA(CREB)-CMV-SaCas9-DIO-pA
FEATURES             Location/Qualifiers
     source          1..7524
                     /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        425..500
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        521..824
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        825..1028
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     979..999
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1025..1049
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(1109..1142)
                     /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(1193..1226)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1277..1324)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1325..4480)
                     /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(4505..4525)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4528..4537
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4559..4592
                     /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    4643..4676
                     /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(4729..4748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4787..4927
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4787..4916
                     /label=AAV2 ITR
     rep_origin      5002..5457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5089..5108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5299..5320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5474..5493)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5593..5615
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5653..5671)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5739..5843
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5844..5912,5913..6704)
                     /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(6062..6081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6875..7463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7364..7383
                     /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 caccggagca gacaaccagc
      421 agaggtttta gtactctgga aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc
      481 tcgtcaactt gttggcgaga tttttacgcg tggagttccg cgttacataa cttacggtaa
      541 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      601 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      661 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      721 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      781 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      841 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      901 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      961 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1021 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     1081 tccatagaag acaccggtcg actccggaat aacttcgtat aggatacttt atacgaagtt
     1141 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
     1201 gtatagcata cattatacga agttatggcg cgccttactt gtgaattcct aggccagcta
     1261 tctagatatc ggatcccttt ttcttttttg cctggccggc ctttttcgtg gccgccggcc
     1321 ttttgccctt tttgatgatc tgagggtgct tcttagattt cacttcatac aggttgccca
     1381 gaatgtctgt gctgtacttc ttaatgctct gggtcttgga ggcgattgtc ttaatgatcc
     1441 tggggggcct cttgtcgttc atgttttcca ggtactcgcg gtaggtgatg tcgatcatgt
     1501 tcacttcgat ccggttcagc aggtcgttgt tcacgccgat cactctatac agctcgccgt
     1561 tgatcttgat cagatcgttg ttgtagaagg aggcgataaa ctcggcctgg ttgctgatct
     1621 tcttcagctt cttagcttcc tcatagcact tgctattcac ttcgtagtag ttttcttttt
     1681 tgatcacatc cagattcttc acggtcacga acttgtacac gccattgtcc aggtacacgt
     1741 cgaatctgta gggcttcagg gacagcttca cgaccttgtt tctgctgttg gggtagtcgt
     1801 cggtgatgtc cagatgggcg ttcagtttgt tgccgtaata cttaatcttc ttgatcacgg
     1861 ggccgttgtc ctttttggag tacttggtca ggtagttccc ggtttcctcg tagtacttgt
     1921 acaggggatt cttctcgtcg ccgtactgtt ccataatcag cttcagtttc tggtaggtct
     1981 gggggtcgtg gtggtacatc agcagctttt cggggctctt gttgatcagc tttttcagct
     2041 tgtcattgtc cttgtcgtac aggccgttca gattgttcac gatcagggtg ttgcccttgt
     2101 cgtccttccg ggtggagtac agggtgtcgt taatcagctc tctattaggc ttcttgtcca
     2161 cccggtggct gtacttgtag tccttgaagt ccttaatgtg cttgatctgg tggggggtga
     2221 tgaagatctc tttgtactcc tgctcggttt cgatctcggg catgctctcg gcctgctttt
     2281 cctcgaacat ctggttttcc atcacttttt tggccttgtc cagtttcttc cactctttga
     2341 agatgaaatc ggcgttggca atgatcaggg cgtcctcggc gtggtgcttg taccccttgt
     2401 tccgctcttt cttaaacttc cacttccgcc gcagaaagct ggtgaagccg ccattgatgg
     2461 acttcacttt cacgtccagg ttgttcactc tgaagtagct ccgcagcagg ttcatcaggc
     2521 ctctggtggc gtatctggta tccaccaggt tccggttgat gaagtctttc tgcacggaga
     2581 acctgttgat gtcccgttct tccagcagat actctttctt ggtcttgctg attctgccct
     2641 tgcccttggc cagattcagg atgtgcttct tgaaggtttc gtagctgatc ttgctgtcgc
     2701 tgctgctcag gtactggaat ggggtccggt tgcccttctt gctgttttct tcctgcttca
     2761 cgagcacctt gttgttgaag ctgttgtcga aggacacgct tctggggatg atgtggtcca
     2821 cctcatagtt gaaggggttg ttcagcagat cttccagagg gatggcttcc aggctgtaca
     2881 ggcacttgcc ttcctgcatg tcgtgcagct tgatcttctc gatcaggtac ttggcgttct
     2941 ctttgccggt ggtccggatg atttcctcga tccgctcgtt ggtctgccgg ttccgcttct
     3001 gcatctcgtt gatcattttc tgggcgtcct tggagttctt ctcgcgggcc agctcgataa
     3061 tgatgtcgtt gggcaggccg tacttcttga tgatggcgtt gatcactttg atgctctgga
     3121 tgaagcttct cttcacgacg gggctcagga tgaagtcgtc caccagggtg gtggggatct
     3181 ctttctgctg ggacaggtcc accttcttgg gcaccagctt cagccggttg aagatagcga
     3241 tctggttgtc gttggtgtgc cacagctcgt ccaggatcag gttgatggcc ttcaggctca
     3301 ggttgtgggt gccggtatag cccttcagat tagagatctg ctcgatctct tcctgggtca
     3361 gctcggagtt cagattggtc agttcttcct ggatgtcctc gctgctctgg tagatggtca
     3421 ggatcttggc aatctgatcc agcagctcgg cgttctcaat aatctctttc cgggcggtaa
     3481 tgtccttgat gtcgtggtac accttcaggt tggtgaactc gggcttgccg gtgctggtca
     3541 ctctgtagcc cttaatatcc tcttcgttca cgaggatttc tttggcgatc tgcttcaggg
     3601 tgggcttctt cttctgcttg aacacgttct cgatgatctg gaacttctcg taatattcca
     3661 gcttctcgtt ctcgtccctg gtgatcacga gattgttcag gtcgttcagg gcgttgtaca
     3721 ggtcggcgtt gtaggcgtac ttcacgctcc gcagttcctc ggggaagtag gtgcagtggc
     3781 ccatcagcat ctcgtaccat tctttgatgt ccttccagcc gaaggggctg ccctcgccag
     3841 gtccctcata gtaggtccgc cgggtttcca gcaggtcgat gtaggtgtcg atgaagctct
     3901 ggtccagctg gtggtaggcc ttctgcacct tcagcagctg tttggcttct ttcacgtagt
     3961 cgctggtctt gaatctgttg atgctgcccc gcacttcgcc gtctttcttc agccgttcca
     4021 gctgcagttc ggccacgtat ttctcttcca gggccttgct gttccggctg atctgctctt
     4081 tggtggacag ctcgttgccg gtgtcctctt ccacctcgtt cacgttgtgc acgcctcttc
     4141 tcttggccag gtgcagcagg gcggcagaga actcttcctc gctcagcttc tggctcaggc
     4201 ccttcactct ggcctcgtag gggttgatgc cgctcagctc gctgtggtcg gtcagcaggt
     4261 tgtagtcgaa cagcagcttc ttcactctct ggattctatg ccgcctccgc cgcttcagcc
     4321 ttctggcgcc tctcttgctc cgcctgccct cgttgttttc cacgttggcc tctttgaaca
     4381 gccgcacgcc ggcatcgatc acgtcccgtg tctcgtagtc gatgatgccg tagcccacgc
     4441 tggtgatgcc gatgtccagg cccaggatgt agttccgctt ggctgctggg actccgtgga
     4501 taccgacctt ccgcttcttc tttggggcca tggtggcacc ggtcatggtg gcgctagcat
     4561 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     4621 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     4681 tcgatactag tctcgagttt attgcagctt ataatggtta caaataaagc aatagcatca
     4741 caaatttcac aaataaagca tttttttcac tggaccgagc ggccgcagga acccctagtg
     4801 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4861 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4921 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4981 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5041 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5101 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5161 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     5221 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5281 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5341 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5401 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5461 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5521 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5581 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5641 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5701 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5761 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5821 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5881 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5941 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6001 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6061 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6121 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6181 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6241 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6301 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6361 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6421 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6481 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6541 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6601 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6661 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6721 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6781 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6841 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6901 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6961 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7021 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7081 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7141 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7201 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7261 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7321 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7381 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7441 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7501 tttgctggcc ttttgctcac atgt
//
