LOCUS       sequence-232036-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible H1 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 6271)
  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 6271)
  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: 
            pJEP324-pAAV-FullH1TO-SaCa9sgRNAi(emx1sg1)-CMV-TetR-2A-EGFP-KASH-WPR
            E-shortPA
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        8..83
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        116..419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        420..623
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     574..594
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     620..644
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             771..1394
                     /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(847..866)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1413..1466
                     /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(1467..1469,1470..2180)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1467..1469,1470..2180)
                     /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(1509..1530)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1770..1789)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2117..2138
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2472..3060
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2525..2545)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2943..2954)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3147..3287
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3147..3276
                     /label=AAV2 ITR
     rep_origin      3362..3817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3449..3468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3659..3680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3834..3853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3953..3975
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4013..4031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4099..4203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4204..4272,4273..5064)
                     /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(4422..4441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5235..5823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5724..5743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5885..6014
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        6159..6253
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     6170..6189
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    6207..6225
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6235..6253
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ctggccagtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt
       61 atctcgtcaa cttgttggcg agatttttac cggtccatgg actagttcta gagtccgtta
      121 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      181 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      241 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      301 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      361 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      421 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      481 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      541 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      601 gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
      661 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc ggccgggaac
      721 ggtgcattgg aacggacctg caggaattga tccgcggccg caccggtacc atgatgtcca
      781 gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg
      841 aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt
      901 ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc
      961 accatactca cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg
     1021 ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta
     1081 cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac
     1141 aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag
     1201 gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta
     1261 ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag
     1321 agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat
     1381 gtgaaagtgg gtccgcggct agcggcagtg gagagggcag aggaagtctg ctaacatgcg
     1441 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     1501 tacccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1561 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1621 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1681 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1741 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1801 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1861 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1921 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1981 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2041 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2101 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2161 gcatggacga gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca
     2221 gcaggatgcc ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa
     2281 gggtgatcag ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct
     2341 gcctgctacc tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat
     2401 ccttctaccc catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcgatatca
     2461 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2521 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2581 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2641 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2701 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2761 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2821 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     2881 cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2941 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3001 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3061 atcgataccg agcgctaata aaagatcttt attttcatta gatctgtgtg ttggtttttt
     3121 gtgtcacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     3181 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3241 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3301 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3361 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3421 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3481 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3541 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3601 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3661 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     3721 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     3781 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     3841 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3901 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3961 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4021 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4081 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4141 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4201 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4261 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4321 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4381 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4441 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4501 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4561 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4621 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4681 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4741 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4801 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4861 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4921 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4981 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5041 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5101 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5161 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5221 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5281 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5341 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5401 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5461 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5521 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5581 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5641 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5701 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5761 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5821 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5881 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     5941 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     6001 cactaggggt tcctgcggcc gcacgcgtga attcgaacgc tgacgtcatc aacccgctcc
     6061 aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag
     6121 gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt
     6181 tctgggaaat caccataaac gtgaaatccc tatcagtgat agagacttat aagttcccta
     6241 tcagtgatag agacaccggc ctccccaaag c
//
