LOCUS       sequence-229517-        6274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible H1 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 6274)
  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 6274)
  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: 
            pJEP325-pAAV-FullH1TO-SaCa9sgRNAi(CREB)-CMV-TetR-2A-EGFP-KASH-WPRE-s
            hortPA
FEATURES             Location/Qualifiers
     source          1..6274
                     /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        279..373
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     290..309
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    355..373
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        398..473
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        506..809
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        810..1013
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     964..984
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1010..1034
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1161..1784
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1237..1256)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1803..1856
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1857..1859,1860..2570)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1857..1859,1860..2570)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1899..1920)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2160..2179)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2507..2528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2862..3450
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2915..2935)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3333..3344)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3537..3677
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3537..3666
                     /label=AAV2 ITR
     rep_origin      3752..4207
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3839..3858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4049..4070
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4224..4243)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4343..4365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4403..4421)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4489..4593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4594..4662,4663..5454)
                     /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(4812..4831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5625..6213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6114..6133
                     /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 tgacgcgtga attcgaacgc tgacgtcatc aacccgctcc
      181 aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag
      241 gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt
      301 tctgggaaat caccataaac gtgaaatccc tatcagtgat agagacttat aagttcccta
      361 tcagtgatag agacaccgga gcagacaacc agcagaggtt ttagtactct ggaaacagaa
      421 tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agatttttac
      481 cggtccatgg actagttcta gagtccgtta cataacttac ggtaaatggc ccgcctggct
      541 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      601 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      661 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      721 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      781 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      841 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      901 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      961 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
     1021 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
     1081 gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacggacctg caggaattga
     1141 tccgcggccg caccggtacc atgatgtcca gattagataa aagtaaagtg attaacagcg
     1201 cattagagct gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga
     1261 agctaggtgt agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg
     1321 acgccttagc cattgagatg ttagataggc accatactca cttttgccct ttagaagggg
     1381 aaagctggca agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc
     1441 atcgcgatgg agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc
     1501 tcgaaaatca attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg
     1561 cactcagcgc tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag
     1621 tcgctaaaga agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag
     1681 ctatcgaatt atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga
     1741 tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg gtccgcggct agcggcagtg
     1801 gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga
     1861 gcaagggcga ggagctgttc accggggtgg tacccatcct ggtcgagctg gacggcgacg
     1921 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1981 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2041 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2101 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2161 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2221 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2281 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2341 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2401 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2461 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2521 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggactca
     2581 gatctcgaga ggaggaggag gagacagaca gcaggatgcc ccacctcgac agccccggca
     2641 gctcccagcc gagacgctcc ttcctctcaa gggtgatcag ggcagcgcta ccgttgcagc
     2701 tgcttctgct gctgctgctg ctcctggcct gcctgctacc tgcctctgaa gatgactaca
     2761 gctgcaccca ggccaacaac tttgcccgat ccttctaccc catgctgcgg tacaccaacg
     2821 ggccacctcc cacctaggaa ttcgatatca agcttatcga taatcaacct ctggattaca
     2881 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     2941 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3001 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3061 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3121 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3181 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3241 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     3301 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3361 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3421 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctaata aaagatcttt
     3481 attttcatta gatctgtgtg ttggtttttt gtgtcacgtg cggaccgagc ggccgcagga
     3541 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3601 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3661 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3721 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3781 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     3841 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3901 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3961 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4021 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4081 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4141 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4201 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4261 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4321 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4381 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4441 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4501 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4561 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4621 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4681 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4741 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4801 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4861 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4921 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4981 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5041 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5101 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5161 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5221 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5281 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5341 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5401 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5461 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5521 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5581 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5641 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5701 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5761 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5821 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5881 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5941 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6001 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6061 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6121 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6181 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6241 ttcctggcct tttgctggcc ttttgctcac atgt
//
