LOCUS       sequence-229556-        5067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 gRNA expression cassette with a gRNA targeting 
            CREB, 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 5067)
  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 5067)
  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: pJEP320-pAAV-U6SaCas9gRNA(CREB3)_EFS-GFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..5067
                     /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        414..489
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        538..749
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      775..784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(781..783,784..786,787..1497)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(781..783,784..786,787..1497)
                     /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(826..847)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1087..1106)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1434..1455
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1814..2290
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2013..2032)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2330..2470
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2330..2459
                     /label=AAV2 ITR
     rep_origin      2545..3000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2632..2651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2842..2863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3017..3036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3136..3158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3196..3214)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3282..3386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3387..3455,3456..4247)
                     /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(3605..3624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4418..5006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4907..4926
                     /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 accggagcag acaaccagca gaggttttag
      421 tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct cgtcaacttg
      481 ttggcgagat ttttaccggt ccatggacta gttcgagtgg ctccggtgcc cgtcagtggg
      541 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaacgg
      601 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      661 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
      721 ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgtctagagt cgacgccacc
      781 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      841 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      901 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      961 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1021 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1081 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1141 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1201 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1261 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1321 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1381 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1441 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
     1501 ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca cctcgacagc
     1561 cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc agcgctaccg
     1621 ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc ctctgaagat
     1681 gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat gctgcggtac
     1741 accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgatac cgagcgctgc
     1801 tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga
     1861 agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc
     1921 tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca
     1981 agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt
     2041 ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca
     2101 gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt
     2161 ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt
     2221 gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt
     2281 ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta
     2341 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     2401 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     2461 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2521 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2581 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     2641 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2701 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2761 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2821 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     2881 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     2941 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     3001 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3061 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3121 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3181 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3241 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3301 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3361 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3421 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3481 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3541 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3601 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3661 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3721 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3781 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3841 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3901 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3961 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4021 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4081 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4141 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4201 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4261 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4321 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4381 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4441 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4501 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4561 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4621 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4681 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4741 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4801 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4861 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4921 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4981 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5041 ccttttgctg gccttttgct cacatgt
//
