LOCUS       sequence-214988-        5077 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 5077)
  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 5077)
  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..5077
                     /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        423..498
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        547..758
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      785..794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(791..793,794..796,797..1507)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(791..793,794..796,797..1507)
                     /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(836..857)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1097..1116)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1444..1465
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1824..2300
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2023..2042)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2340..2480
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2340..2469
                     /label=AAV2 ITR
     rep_origin      2555..3010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2642..2661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2852..2873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3027..3046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3146..3168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3206..3224)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3292..3396
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3397..3465,3466..4257)
                     /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(3615..3634)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4428..5016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4917..4936
                     /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 caccggaaga gcgagctctt
      421 ctgttttagt actctggaaa cagaatctac taaaacaagg caaaatgccg tgtttatctc
      481 gtcaacttgt tggcgagatt tttaccggtc catggactag ttcgagtggc tccggtgccc
      541 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
      601 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
      661 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
      721 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc ggtctagagt
      781 cgacgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      841 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      901 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      961 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1021 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1081 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1141 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1201 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1261 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1321 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1381 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1441 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1501 gtacaagtcc ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca
     1561 cctcgacagc cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc
     1621 agcgctaccg ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc
     1681 ctctgaagat gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat
     1741 gctgcggtac accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgatac
     1801 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     1861 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     1921 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     1981 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     2041 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     2101 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     2161 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     2221 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     2281 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     2341 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     2401 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     2461 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2521 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     2581 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2641 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2701 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2761 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2821 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2881 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2941 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3001 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3061 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3121 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3181 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3241 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3301 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3361 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3421 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3481 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3541 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3601 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3661 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3721 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3781 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3841 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3901 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3961 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4021 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4081 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4141 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4201 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4261 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4321 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4381 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4501 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4561 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4621 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4681 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4741 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4801 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4861 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4921 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4981 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5041 cggttcctgg ccttttgctg gccttttgct cacatgt
//
