LOCUS       sequence-214989-        5080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 gRNA expression cassette with a gRNA targeting 
            EMX1, followed by an EFS driven GFP-KASH in a separate reading 
            frame..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5080)
  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 5080)
  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: pJEP318-pAAV-U6SaCas9gRNA(emx1sg1)-EFS-GFP-KAS...
FEATURES             Location/Qualifiers
     source          1..5080
                     /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        426..501
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        550..761
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      788..797
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(794..796,797..799,800..1510)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(794..796,797..799,800..1510)
                     /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(839..860)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1100..1119)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1447..1468
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1827..2303
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2026..2045)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2343..2483
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2343..2472
                     /label=AAV2 ITR
     rep_origin      2558..3013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2645..2664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2855..2876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3030..3049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3149..3171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3209..3227)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3295..3399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3400..3468,3469..4260)
                     /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(3618..3637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4431..5019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4920..4939
                     /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 caccggcctc cccaaagcct
      421 ggccagtttt agtactctgg aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat
      481 ctcgtcaact tgttggcgag atttttaccg gtccatggac tagttcgagt ggctccggtg
      541 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      601 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      661 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      721 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcggtctag
      781 agtcgacgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      841 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      901 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      961 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1021 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1081 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1141 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1201 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1261 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1321 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1381 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1441 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1501 gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca gcaggatgcc
     1561 ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa gggtgatcag
     1621 ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct gcctgctacc
     1681 tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat ccttctaccc
     1741 catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcgatatca agcttatcga
     1801 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     1861 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     1921 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     1981 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     2041 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     2101 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     2161 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     2221 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     2281 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     2341 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     2401 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     2461 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     2521 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     2581 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2641 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2701 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2761 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2821 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2881 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     2941 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3001 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3061 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3121 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3181 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3241 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3301 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3361 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3421 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3481 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3541 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3601 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3661 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3721 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3781 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3841 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3901 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3961 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4021 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4081 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4141 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4201 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4261 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4321 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4381 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4441 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4501 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4561 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4621 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4681 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4741 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4801 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4861 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4921 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4981 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5041 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
