LOCUS       sequence-229555-        5065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 gRNA expression cassette without a gRNA. Followed 
            by an EFS driven GFP-KASH in a separate reading frame. SapI can be 
            used to clone in gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5065)
  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 5065)
  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: pJEP317-pAAV-U6SaCas9gRNA(SapI)-EFS-GFP- KASH-pA
FEATURES             Location/Qualifiers
     source          1..5065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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"
     promoter        536..747
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      773..782
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(779..781,782..784,785..1495)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(779..781,782..784,785..1495)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(824..845)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1085..1104)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1432..1453
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1812..2288
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2011..2030)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2328..2468
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2328..2457
                     /label=AAV2 ITR
     rep_origin      2543..2998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2630..2649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2840..2861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3015..3034)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3134..3156
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3194..3212)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3280..3384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3385..3453,3454..4245)
                     /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(3603..3622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4416..5004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4905..4924
                     /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 ttaccggtcc atggactagt tcgagtggct ccggtgcccg tcagtgggca
      541 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt
      601 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      661 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      721 cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg tctagagtcg acgccaccat
      781 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      841 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      901 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      961 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1021 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1081 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1141 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1201 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1261 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1321 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1381 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1441 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     1501 actcagatct cgagaggagg aggaggagac agacagcagg atgccccacc tcgacagccc
     1561 cggcagctcc cagccgagac gctccttcct ctcaagggtg atcagggcag cgctaccgtt
     1621 gcagctgctt ctgctgctgc tgctgctcct ggcctgcctg ctacctgcct ctgaagatga
     1681 ctacagctgc acccaggcca acaactttgc ccgatccttc taccccatgc tgcggtacac
     1741 caacgggcca cctcccacct aggaattcga tatcaagctt atcgataccg agcgctgctc
     1801 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     1861 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     1921 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     1981 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     2041 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     2101 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     2161 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     2221 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     2281 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     2341 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     2401 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     2461 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2521 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2581 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     2641 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2701 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     2761 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2821 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     2881 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     2941 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     3001 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3061 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3121 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3181 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3241 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3301 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3361 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3421 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3481 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3541 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3601 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3661 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3721 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3781 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3841 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3901 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3961 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4021 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4081 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4141 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4201 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4261 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4321 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4381 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4441 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4501 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4561 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4621 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4681 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4741 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4801 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4861 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4921 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4981 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5041 ttttgctggc cttttgctca catgt
//
