LOCUS       sequence-229553-        5068 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 5068)
  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 5068)
  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..5068
                     /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        415..490
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        539..750
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      776..785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(782..784,785..787,788..1498)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(782..784,785..787,788..1498)
                     /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(827..848)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1088..1107)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1435..1456
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1815..2291
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2014..2033)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2331..2471
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2331..2460
                     /label=AAV2 ITR
     rep_origin      2546..3001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2843..2864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3018..3037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3137..3159
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3197..3215)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3283..3387
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3388..3456,3457..4248)
                     /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(3606..3625)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4419..5007
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4908..4927
                     /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 accggcctcc ccaaagcctg gccagtttta
      421 gtactctgga aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt
      481 gttggcgaga tttttaccgg tccatggact agttcgagtg gctccggtgc ccgtcagtgg
      541 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg
      601 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      661 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
      721 tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgtctagag tcgacgccac
      781 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      841 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      901 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      961 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1021 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1081 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1141 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1201 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1261 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1321 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1381 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1441 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc
     1501 cggactcaga tctcgagagg aggaggagga gacagacagc aggatgcccc acctcgacag
     1561 ccccggcagc tcccagccga gacgctcctt cctctcaagg gtgatcaggg cagcgctacc
     1621 gttgcagctg cttctgctgc tgctgctgct cctggcctgc ctgctacctg cctctgaaga
     1681 tgactacagc tgcacccagg ccaacaactt tgcccgatcc ttctacccca tgctgcggta
     1741 caccaacggg ccacctccca cctaggaatt cgatatcaag cttatcgata ccgagcgctg
     1801 ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     1861 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     1921 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     1981 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     2041 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     2101 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     2161 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     2221 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     2281 tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct
     2341 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     2401 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     2461 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2521 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     2641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2761 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     2881 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     2941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     3001 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3061 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3121 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3181 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3241 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3301 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3361 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3421 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3481 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3541 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3601 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3661 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3721 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3781 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3841 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3901 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3961 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4021 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4081 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4141 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4201 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4261 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4321 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4381 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4441 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4501 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4561 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4621 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4681 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4741 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4801 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4861 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4921 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4981 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5041 gccttttgct ggccttttgc tcacatgt
//
