LOCUS       Exported                7511 bp ds-DNA     circular SYN 12-MAY-2021
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    pJEP314-pAAV-U6SaCas9gRNA(SapI)-CMV-SaCas9-DIO-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7511)
  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 7511)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7511
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..385
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        508..811
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        812..1015
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     966..986
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1012..1036
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1096..1129
                     /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    1180..1213
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1264..1311)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1312..4467)
                     /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(4492..4512)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4515..4524
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4546..4579)
                     /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(4630..4663)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4716..4735)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4774..4914
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4774..4903
                     /label=AAV2 ITR
     rep_origin      4989..5444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5076..5095)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5286..5307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5461..5480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5580..5602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5640..5658)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5726..5830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5831..6691
                     /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(6049..6068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6862..7450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7351..7370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagta
      421 ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt
      481 ggcgagattt ttacgcgtgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      541 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      601 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      661 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      721 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      781 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      841 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      901 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      961 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
     1021 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     1081 ccggtcgact ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta
     1141 gctggattgt agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat
     1201 tatacgaagt tatggcgcgc cttacttgtg aattcctagg ccagctatct agatatcgga
     1261 tccctttttc ttttttgcct ggccggcctt tttcgtggcc gccggccttt tgcccttttt
     1321 gatgatctga gggtgcttct tagatttcac ttcatacagg ttgcccagaa tgtctgtgct
     1381 gtacttctta atgctctggg tcttggaggc gattgtctta atgatcctgg ggggcctctt
     1441 gtcgttcatg ttttccaggt actcgcggta ggtgatgtcg atcatgttca cttcgatccg
     1501 gttcagcagg tcgttgttca cgccgatcac tctatacagc tcgccgttga tcttgatcag
     1561 atcgttgttg tagaaggagg cgataaactc ggcctggttg ctgatcttct tcagcttctt
     1621 agcttcctca tagcacttgc tattcacttc gtagtagttt tcttttttga tcacatccag
     1681 attcttcacg gtcacgaact tgtacacgcc attgtccagg tacacgtcga atctgtaggg
     1741 cttcagggac agcttcacga ccttgtttct gctgttgggg tagtcgtcgg tgatgtccag
     1801 atgggcgttc agtttgttgc cgtaatactt aatcttcttg atcacggggc cgttgtcctt
     1861 tttggagtac ttggtcaggt agttcccggt ttcctcgtag tacttgtaca ggggattctt
     1921 ctcgtcgccg tactgttcca taatcagctt cagtttctgg taggtctggg ggtcgtggtg
     1981 gtacatcagc agcttttcgg ggctcttgtt gatcagcttt ttcagcttgt cattgtcctt
     2041 gtcgtacagg ccgttcagat tgttcacgat cagggtgttg cccttgtcgt ccttccgggt
     2101 ggagtacagg gtgtcgttaa tcagctctct attaggcttc ttgtccaccc ggtggctgta
     2161 cttgtagtcc ttgaagtcct taatgtgctt gatctggtgg ggggtgatga agatctcttt
     2221 gtactcctgc tcggtttcga tctcgggcat gctctcggcc tgcttttcct cgaacatctg
     2281 gttttccatc acttttttgg ccttgtccag tttcttccac tctttgaaga tgaaatcggc
     2341 gttggcaatg atcagggcgt cctcggcgtg gtgcttgtac cccttgttcc gctctttctt
     2401 aaacttccac ttccgccgca gaaagctggt gaagccgcca ttgatggact tcactttcac
     2461 gtccaggttg ttcactctga agtagctccg cagcaggttc atcaggcctc tggtggcgta
     2521 tctggtatcc accaggttcc ggttgatgaa gtctttctgc acggagaacc tgttgatgtc
     2581 ccgttcttcc agcagatact ctttcttggt cttgctgatt ctgcccttgc ccttggccag
     2641 attcaggatg tgcttcttga aggtttcgta gctgatcttg ctgtcgctgc tgctcaggta
     2701 ctggaatggg gtccggttgc ccttcttgct gttttcttcc tgcttcacga gcaccttgtt
     2761 gttgaagctg ttgtcgaagg acacgcttct ggggatgatg tggtccacct catagttgaa
     2821 ggggttgttc agcagatctt ccagagggat ggcttccagg ctgtacaggc acttgccttc
     2881 ctgcatgtcg tgcagcttga tcttctcgat caggtacttg gcgttctctt tgccggtggt
     2941 ccggatgatt tcctcgatcc gctcgttggt ctgccggttc cgcttctgca tctcgttgat
     3001 cattttctgg gcgtccttgg agttcttctc gcgggccagc tcgataatga tgtcgttggg
     3061 caggccgtac ttcttgatga tggcgttgat cactttgatg ctctggatga agcttctctt
     3121 cacgacgggg ctcaggatga agtcgtccac cagggtggtg gggatctctt tctgctggga
     3181 caggtccacc ttcttgggca ccagcttcag ccggttgaag atagcgatct ggttgtcgtt
     3241 ggtgtgccac agctcgtcca ggatcaggtt gatggccttc aggctcaggt tgtgggtgcc
     3301 ggtatagccc ttcagattag agatctgctc gatctcttcc tgggtcagct cggagttcag
     3361 attggtcagt tcttcctgga tgtcctcgct gctctggtag atggtcagga tcttggcaat
     3421 ctgatccagc agctcggcgt tctcaataat ctctttccgg gcggtaatgt ccttgatgtc
     3481 gtggtacacc ttcaggttgg tgaactcggg cttgccggtg ctggtcactc tgtagccctt
     3541 aatatcctct tcgttcacga ggatttcttt ggcgatctgc ttcagggtgg gcttcttctt
     3601 ctgcttgaac acgttctcga tgatctggaa cttctcgtaa tattccagct tctcgttctc
     3661 gtccctggtg atcacgagat tgttcaggtc gttcagggcg ttgtacaggt cggcgttgta
     3721 ggcgtacttc acgctccgca gttcctcggg gaagtaggtg cagtggccca tcagcatctc
     3781 gtaccattct ttgatgtcct tccagccgaa ggggctgccc tcgccaggtc cctcatagta
     3841 ggtccgccgg gtttccagca ggtcgatgta ggtgtcgatg aagctctggt ccagctggtg
     3901 gtaggccttc tgcaccttca gcagctgttt ggcttctttc acgtagtcgc tggtcttgaa
     3961 tctgttgatg ctgccccgca cttcgccgtc tttcttcagc cgttccagct gcagttcggc
     4021 cacgtatttc tcttccaggg ccttgctgtt ccggctgatc tgctctttgg tggacagctc
     4081 gttgccggtg tcctcttcca cctcgttcac gttgtgcacg cctcttctct tggccaggtg
     4141 cagcagggcg gcagagaact cttcctcgct cagcttctgg ctcaggccct tcactctggc
     4201 ctcgtagggg ttgatgccgc tcagctcgct gtggtcggtc agcaggttgt agtcgaacag
     4261 cagcttcttc actctctgga ttctatgccg cctccgccgc ttcagccttc tggcgcctct
     4321 cttgctccgc ctgccctcgt tgttttccac gttggcctct ttgaacagcc gcacgccggc
     4381 atcgatcacg tcccgtgtct cgtagtcgat gatgccgtag cccacgctgg tgatgccgat
     4441 gtccaggccc aggatgtagt tccgcttggc tgctgggact ccgtggatac cgaccttccg
     4501 cttcttcttt ggggccatgg tggcaccggt catggtggcg ctagcataac ttcgtataaa
     4561 gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg
     4621 gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg atactagtct
     4681 cgagtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4741 taaagcattt ttttcactgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     4801 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4861 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4921 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4981 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5041 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     5101 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5161 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     5221 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5281 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     5341 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     5401 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     5461 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     5521 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5581 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     5641 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5701 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5761 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5821 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5881 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5941 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6001 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6061 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6121 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6181 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6241 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6301 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6361 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6421 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6481 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6541 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6601 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6661 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6721 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6781 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6841 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6901 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6961 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7021 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7081 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7141 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7201 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7261 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7321 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7381 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7441 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7501 tgctcacatg t
//
