LOCUS       Exported                7513 bp ds-DNA     circular SYN 12-MAY-2021
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    pJEP315-pAAV-U6SaCas9gRNA(CREB)-CMV-SaCas9-DIO-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7513)
  AUTHORS   Kumar N, Stanford W, Aradhana CS, 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) 13
REFERENCE   2  (bases 1 to 7513)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7513
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     64..83
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        162..237
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        258..561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        562..765
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     716..736
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     762..786
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    846..879
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    930..963
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1014..1061)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1062..4217)
                     /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(4242..4262)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4265..4274
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4296..4329)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(4380..4413)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4466..4485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4524..4664
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4524..4653
                     /label=AAV2 ITR
     rep_origin      4739..5194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4826..4845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5036..5057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5211..5230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5330..5352
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5390..5408)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5476..5580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5581..6441
                     /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(5799..5818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6612..7200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7101..7120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7262..7391
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        7406..133
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     7406..7426
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
       61 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      121 ttgtggaaag gacgaaacac cggagcagac aaccagcaga ggttttagta ctctggaaac
      181 agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt
      241 ttacgcgtgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      301 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      361 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      421 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      481 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      541 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      601 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      661 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      721 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
      781 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccggtcgact
      841 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
      901 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
      961 tatggcgcgc cttacttgtg aattcctagg ccagctatct agatatcgga tccctttttc
     1021 ttttttgcct ggccggcctt tttcgtggcc gccggccttt tgcccttttt gatgatctga
     1081 gggtgcttct tagatttcac ttcatacagg ttgcccagaa tgtctgtgct gtacttctta
     1141 atgctctggg tcttggaggc gattgtctta atgatcctgg ggggcctctt gtcgttcatg
     1201 ttttccaggt actcgcggta ggtgatgtcg atcatgttca cttcgatccg gttcagcagg
     1261 tcgttgttca cgccgatcac tctatacagc tcgccgttga tcttgatcag atcgttgttg
     1321 tagaaggagg cgataaactc ggcctggttg ctgatcttct tcagcttctt agcttcctca
     1381 tagcacttgc tattcacttc gtagtagttt tcttttttga tcacatccag attcttcacg
     1441 gtcacgaact tgtacacgcc attgtccagg tacacgtcga atctgtaggg cttcagggac
     1501 agcttcacga ccttgtttct gctgttgggg tagtcgtcgg tgatgtccag atgggcgttc
     1561 agtttgttgc cgtaatactt aatcttcttg atcacggggc cgttgtcctt tttggagtac
     1621 ttggtcaggt agttcccggt ttcctcgtag tacttgtaca ggggattctt ctcgtcgccg
     1681 tactgttcca taatcagctt cagtttctgg taggtctggg ggtcgtggtg gtacatcagc
     1741 agcttttcgg ggctcttgtt gatcagcttt ttcagcttgt cattgtcctt gtcgtacagg
     1801 ccgttcagat tgttcacgat cagggtgttg cccttgtcgt ccttccgggt ggagtacagg
     1861 gtgtcgttaa tcagctctct attaggcttc ttgtccaccc ggtggctgta cttgtagtcc
     1921 ttgaagtcct taatgtgctt gatctggtgg ggggtgatga agatctcttt gtactcctgc
     1981 tcggtttcga tctcgggcat gctctcggcc tgcttttcct cgaacatctg gttttccatc
     2041 acttttttgg ccttgtccag tttcttccac tctttgaaga tgaaatcggc gttggcaatg
     2101 atcagggcgt cctcggcgtg gtgcttgtac cccttgttcc gctctttctt aaacttccac
     2161 ttccgccgca gaaagctggt gaagccgcca ttgatggact tcactttcac gtccaggttg
     2221 ttcactctga agtagctccg cagcaggttc atcaggcctc tggtggcgta tctggtatcc
     2281 accaggttcc ggttgatgaa gtctttctgc acggagaacc tgttgatgtc ccgttcttcc
     2341 agcagatact ctttcttggt cttgctgatt ctgcccttgc ccttggccag attcaggatg
     2401 tgcttcttga aggtttcgta gctgatcttg ctgtcgctgc tgctcaggta ctggaatggg
     2461 gtccggttgc ccttcttgct gttttcttcc tgcttcacga gcaccttgtt gttgaagctg
     2521 ttgtcgaagg acacgcttct ggggatgatg tggtccacct catagttgaa ggggttgttc
     2581 agcagatctt ccagagggat ggcttccagg ctgtacaggc acttgccttc ctgcatgtcg
     2641 tgcagcttga tcttctcgat caggtacttg gcgttctctt tgccggtggt ccggatgatt
     2701 tcctcgatcc gctcgttggt ctgccggttc cgcttctgca tctcgttgat cattttctgg
     2761 gcgtccttgg agttcttctc gcgggccagc tcgataatga tgtcgttggg caggccgtac
     2821 ttcttgatga tggcgttgat cactttgatg ctctggatga agcttctctt cacgacgggg
     2881 ctcaggatga agtcgtccac cagggtggtg gggatctctt tctgctggga caggtccacc
     2941 ttcttgggca ccagcttcag ccggttgaag atagcgatct ggttgtcgtt ggtgtgccac
     3001 agctcgtcca ggatcaggtt gatggccttc aggctcaggt tgtgggtgcc ggtatagccc
     3061 ttcagattag agatctgctc gatctcttcc tgggtcagct cggagttcag attggtcagt
     3121 tcttcctgga tgtcctcgct gctctggtag atggtcagga tcttggcaat ctgatccagc
     3181 agctcggcgt tctcaataat ctctttccgg gcggtaatgt ccttgatgtc gtggtacacc
     3241 ttcaggttgg tgaactcggg cttgccggtg ctggtcactc tgtagccctt aatatcctct
     3301 tcgttcacga ggatttcttt ggcgatctgc ttcagggtgg gcttcttctt ctgcttgaac
     3361 acgttctcga tgatctggaa cttctcgtaa tattccagct tctcgttctc gtccctggtg
     3421 atcacgagat tgttcaggtc gttcagggcg ttgtacaggt cggcgttgta ggcgtacttc
     3481 acgctccgca gttcctcggg gaagtaggtg cagtggccca tcagcatctc gtaccattct
     3541 ttgatgtcct tccagccgaa ggggctgccc tcgccaggtc cctcatagta ggtccgccgg
     3601 gtttccagca ggtcgatgta ggtgtcgatg aagctctggt ccagctggtg gtaggccttc
     3661 tgcaccttca gcagctgttt ggcttctttc acgtagtcgc tggtcttgaa tctgttgatg
     3721 ctgccccgca cttcgccgtc tttcttcagc cgttccagct gcagttcggc cacgtatttc
     3781 tcttccaggg ccttgctgtt ccggctgatc tgctctttgg tggacagctc gttgccggtg
     3841 tcctcttcca cctcgttcac gttgtgcacg cctcttctct tggccaggtg cagcagggcg
     3901 gcagagaact cttcctcgct cagcttctgg ctcaggccct tcactctggc ctcgtagggg
     3961 ttgatgccgc tcagctcgct gtggtcggtc agcaggttgt agtcgaacag cagcttcttc
     4021 actctctgga ttctatgccg cctccgccgc ttcagccttc tggcgcctct cttgctccgc
     4081 ctgccctcgt tgttttccac gttggcctct ttgaacagcc gcacgccggc atcgatcacg
     4141 tcccgtgtct cgtagtcgat gatgccgtag cccacgctgg tgatgccgat gtccaggccc
     4201 aggatgtagt tccgcttggc tgctgggact ccgtggatac cgaccttccg cttcttcttt
     4261 ggggccatgg tggcaccggt catggtggcg ctagcataac ttcgtataaa gtatcctata
     4321 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     4381 taacttcgta taatgtatgc tatacgaagt tatgaattcg atactagtct cgagtttatt
     4441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4501 ttttcactgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4561 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4621 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     4681 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4741 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4801 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4861 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4921 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4981 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5041 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     5101 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5161 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     5221 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5281 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5341 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5401 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5461 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5521 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5581 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5641 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5701 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5761 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5821 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5881 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5941 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6001 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6061 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6121 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6181 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6241 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6301 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6361 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6421 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6481 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6541 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6601 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6661 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6721 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6781 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6841 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6901 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6961 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7021 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7081 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7141 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7201 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7261 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
     7321 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     7381 taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt catatttgca
     7441 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     7501 attagtacaa aat
//
