LOCUS       sequence-229552-        5069 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 5069)
  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 5069)
  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: pJEP319-pAAV-U6SaCas9gRNA(emx1sg2)-EFS-GFP-KAS...
FEATURES             Location/Qualifiers
     source          1..5069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        123..371
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     123..143
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     294..313
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        394..469
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        518..729
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      755..764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(761..763,764..766,767..1477)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(761..763,764..766,767..1477)
                     /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(806..827)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1067..1086)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1414..1435
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1794..2270
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1993..2012)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2310..2450
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2310..2439
                     /label=AAV2 ITR
     rep_origin      2525..2980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2612..2631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2822..2843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2997..3016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3116..3138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3176..3194)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3262..3366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3367..3435,3436..4227)
                     /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(3585..3604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4398..4986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4887..4906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5048..5069,1..108)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
       61 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
      121 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      181 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      241 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      301 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      361 gacgaaacac cgtggccagg ctttggggag gccgttttag tactctggaa acagaatcta
      421 ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat ttttaccggt
      481 ccatggacta gttcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      541 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
      601 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      661 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
      721 agaacacagg tgtcgtgacg cgtctagagt cgacgccacc atggtgagca agggcgagga
      781 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      841 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      901 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
      961 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1021 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1081 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1141 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1201 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1261 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1321 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1381 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1441 cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat ctcgagagga
     1501 ggaggaggag acagacagca ggatgcccca cctcgacagc cccggcagct cccagccgag
     1561 acgctccttc ctctcaaggg tgatcagggc agcgctaccg ttgcagctgc ttctgctgct
     1621 gctgctgctc ctggcctgcc tgctacctgc ctctgaagat gactacagct gcacccaggc
     1681 caacaacttt gcccgatcct tctaccccat gctgcggtac accaacgggc cacctcccac
     1741 ctaggaattc gatatcaagc ttatcgatac cgagcgctgc tcgagagatc tacgggtggc
     1801 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     1861 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     1921 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     1981 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     2041 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     2101 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     2161 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     2221 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt
     2281 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     2341 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     2401 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     2461 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     2521 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2581 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     2641 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2701 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     2761 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2821 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     2881 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     2941 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     3001 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     3061 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3121 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     3181 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3241 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3301 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3361 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3421 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3481 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3541 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3601 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3661 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3721 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3781 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3841 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3901 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3961 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4021 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4081 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4141 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4201 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5041 cacatgtcct gcaggcagct gcgcgctcg
//
