LOCUS       Exported                7021 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An EFS driven inverted de-catalyzed SaCas9 fused to the 
            transcription activator VP64. dSaCas9-VP64 is floxed to render the 
            system cre-dependent..
ACCESSION   .
VERSION     .
KEYWORDS    pJEP308-pAAV-EFS-dSaCas9-VP64-Dio-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7021)
  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 7021)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7021
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        181..392
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    427..460
                     /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    511..544
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(594..743)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(774..821)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4002..4022)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4025..4034
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4056..4089)
                     /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(4140..4173)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4226..4245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4284..4424
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4284..4413
                     /label=AAV2 ITR
     rep_origin      4499..4954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4586..4605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4796..4817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4971..4990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5090..5112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5150..5168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5236..5340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5341..6201
                     /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(5559..5578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6372..6960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6861..6880
                     /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 cgcgttcgag tggctccggt gcccgtcagt
      181 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
      241 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      301 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      361 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggaat tcgagtcgac
      421 tccggaataa cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg
      481 tagctgctat tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag
      541 ttatggcgcg ccttacttgt gaattcggta cctctagatc atcttgagtt aatcagcatg
      601 tccaggtcga aatcatcaag ggcgtcactg ccgagcatgt cgaggtcaaa gtcatcaagg
      661 gcatccgaac caagcatgtc aaggtcaaaa tcatcgaggg cgtcacttcc cagcatatcc
      721 agatcaaaat cgtccaatgc gtcagcccgt ccggaaccgc tggcctcgga tccctttttc
      781 ttttttgcct ggccggcctt tttcgtggcc gccggccttt tgcccttttt gatgatctga
      841 gggtgcttct tagatttcac ttcatacagg ttgcccagaa tgtctgtgct gtacttctta
      901 atgctctggg tcttggaggc gattgtctta atgatcctgg ggggcctctt gtcgttcatg
      961 ttttccaggt actcgcggta ggtgatgtcg atcatgttca cttcgatccg gttcagcagg
     1021 tcgttgttca cgccgatcac tctatacagc tcgccgttga tcttgatcag atcgttgttg
     1081 tagaaggagg cgataaactc ggcctggttg ctgatcttct tcagcttctt agcttcctca
     1141 tagcacttgc tattcacttc gtagtagttt tcttttttga tcacatccag attcttcacg
     1201 gtcacgaact tgtacacgcc attgtccagg tacacgtcga atctgtaggg cttcagggac
     1261 agcttcacga ccttgtttct gctgttgggg tagtcgtcgg tgatgtccag atgggcgttc
     1321 agtttgttgc cgtaatactt aatcttcttg atcacggggc cgttgtcctt tttggagtac
     1381 ttggtcaggt agttcccggt ttcctcgtag tacttgtaca ggggattctt ctcgtcgccg
     1441 tactgttcca taatcagctt cagtttctgg taggtctggg ggtcgtggtg gtacatcagc
     1501 agcttttcgg ggctcttgtt gatcagcttt ttcagcttgt cattgtcctt gtcgtacagg
     1561 ccgttcagat tgttcacgat cagggtgttg cccttgtcgt ccttccgggt ggagtacagg
     1621 gtgtcgttaa tcagctctct attaggcttc ttgtccaccc ggtggctgta cttgtagtcc
     1681 ttgaagtcct taatgtgctt gatctggtgg ggggtgatga agatctcttt gtactcctgc
     1741 tcggtttcga tctcgggcat gctctcggcc tgcttttcct cgaacatctg gttttccatc
     1801 acttttttgg ccttgtccag tttcttccac tctttgaaga tgaaatcggc gttggcaatg
     1861 atcagggcgt cctcggcgtg gtgcttgtac cccttgttcc gctctttctt aaacttccac
     1921 ttccgccgca gaaagctggt gaagccgcca ttgatggact tcactttcac gtccaggttg
     1981 ttcactctga agtagctccg cagcaggttc atcaggcctc tggtggcgta tctggtatcc
     2041 accaggttcc ggttgatgaa gtctttctgc acggagaacc tgttgatgtc ccgttcttcc
     2101 agcagatact ctttcttggt cttgctgatt ctgcccttgc ccttggccag attcaggatg
     2161 tgcttcttga aggtttcgta gctgatcttg ctgtcgctgc tgctcaggta ctggaatggg
     2221 gtccggttgc ccttcttgct ggcttcttcc tgcttcacga gcaccttgtt gttgaagctg
     2281 ttgtcgaagg acacgcttct ggggatgatg tggtccacct catagttgaa ggggttgttc
     2341 agcagatctt ccagagggat ggcttccagg ctgtacaggc acttgccttc ctgcatgtcg
     2401 tgcagcttga tcttctcgat caggtacttg gcgttctctt tgccggtggt ccggatgatt
     2461 tcctcgatcc gctcgttggt ctgccggttc cgcttctgca tctcgttgat cattttctgg
     2521 gcgtccttgg agttcttctc gcgggccagc tcgataatga tgtcgttggg caggccgtac
     2581 ttcttgatga tggcgttgat cactttgatg ctctggatga agcttctctt cacgacgggg
     2641 ctcaggatga agtcgtccac cagggtggtg gggatctctt tctgctggga caggtccacc
     2701 ttcttgggca ccagcttcag ccggttgaag atagcgatct ggttgtcgtt ggtgtgccac
     2761 agctcgtcca ggatcaggtt gatggccttc aggctcaggt tgtgggtgcc ggtatagccc
     2821 ttcagattag agatctgctc gatctcttcc tgggtcagct cggagttcag attggtcagt
     2881 tcttcctgga tgtcctcgct gctctggtag atggtcagga tcttggcaat ctgatccagc
     2941 agctcggcgt tctcaataat ctctttccgg gcggtaatgt ccttgatgtc gtggtacacc
     3001 ttcaggttgg tgaactcggg cttgccggtg ctggtcactc tgtagccctt aatatcctct
     3061 tcgttcacga ggatttcttt ggcgatctgc ttcagggtgg gcttcttctt ctgcttgaac
     3121 acgttctcga tgatctggaa cttctcgtaa tattccagct tctcgttctc gtccctggtg
     3181 atcacgagat tgttcaggtc gttcagggcg ttgtacaggt cggcgttgta ggcgtacttc
     3241 acgctccgca gttcctcggg gaagtaggtg cagtggccca tcagcatctc gtaccattct
     3301 ttgatgtcct tccagccgaa ggggctgccc tcgccaggtc cctcatagta ggtccgccgg
     3361 gtttccagca ggtcgatgta ggtgtcgatg aagctctggt ccagctggtg gtaggccttc
     3421 tgcaccttca gcagctgttt ggcttctttc acgtagtcgc tggtcttgaa tctgttgatg
     3481 ctgccccgca cttcgccgtc tttcttcagc cgttccagct gcagttcggc cacgtatttc
     3541 tcttccaggg ccttgctgtt ccggctgatc tgctctttgg tggacagctc gttgccggtg
     3601 tcctcttcca cctcgttcac gttgtgcacg cctcttctct tggccaggtg cagcagggcg
     3661 gcagagaact cttcctcgct cagcttctgg ctcaggccct tcactctggc ctcgtagggg
     3721 ttgatgccgc tcagctcgct gtggtcggtc agcaggttgt agtcgaacag cagcttcttc
     3781 actctctgga ttctatgccg cctccgccgc ttcagccttc tggcgcctct cttgctccgc
     3841 ctgccctcgt tgttttccac gttggcctct ttgaacagcc gcacgccggc atcgatcacg
     3901 tcccgtgtct cgtagtcgat gatgccgtag cccacgctgg tgatgccgat ggccaggccc
     3961 aggatgtagt tccgcttggc tgctgggact ccgtggatac cgaccttccg cttcttcttt
     4021 ggggccatgg tggcaccggt catggtggcg ctagcataac ttcgtataaa gtatcctata
     4081 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     4141 taacttcgta taatgtatgc tatacgaagt tatgaattcg atactagtct cgagtttatt
     4201 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4261 ttttcactgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4321 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4381 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     4441 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4501 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4561 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4621 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4681 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4741 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4801 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     4861 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4921 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4981 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5041 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5101 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5161 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5221 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5341 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5401 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5461 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5521 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5581 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5641 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5701 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5761 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5821 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5881 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5941 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6001 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6061 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6121 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6181 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6241 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6301 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6361 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6421 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6481 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6541 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6601 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6661 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6721 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6781 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6841 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6901 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6961 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7021 t
//
