LOCUS       sequence-214994-        6297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible H1 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 a gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6297)
  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 6297)
  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: 
            pJEP323-pAAV-FullH1TO-SaCa9gRNAi(SapI)-CMV-TetR-2A-EGFP-KASH-WPRE-sh
            ortPA Empty Cassette
FEATURES             Location/Qualifiers
     source          1..6297
                     /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
     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"
     protein_bind    357..375
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    385..403
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        424..499
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        532..835
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        836..1039
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     990..1010
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1036..1060
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1184..1807
                     /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(1260..1279)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1826..1879
                     /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(1880..1882,1883..2593)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1880..1882,1883..2593)
                     /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(1922..1943)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2183..2202)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2530..2551
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2885..3473
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2938..2958)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3356..3367)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3560..3700
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3560..3689
                     /label=AAV2 ITR
     rep_origin      3775..4230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3862..3881)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4072..4093
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4247..4266)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4366..4388
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4426..4444)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4512..4616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4617..4685,4686..5477)
                     /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(4835..4854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5648..6236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6137..6156
                     /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 tgaaactccc
      361 tatcagtgat agagattata tatctcccta tcagtgatag agaccggaag agcgagctct
      421 tctgttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct
      481 cgtcaacttg ttggcgagat ttttaccggt ccatggacta gttctagagt ccgttacata
      541 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      601 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      661 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      721 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      781 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      841 gcggttttgg cagtacatca atgggcgtga atagcggttt gactcacggg gatttccaag
      901 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      961 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1021 ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg
     1081 ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc gggaacggtg
     1141 cattggaacg ctgcaggaat tgatccgcgg ccgcaccggt accatgatgt ccagattaga
     1201 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt
     1261 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt
     1321 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac
     1381 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag
     1441 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc
     1501 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt
     1561 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt
     1621 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag
     1681 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc
     1741 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag
     1801 tgggtccgcg gctagcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     1861 cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1921 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1981 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2041 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2101 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2161 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2221 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2281 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2341 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2401 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2461 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2521 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2581 cgagctgtac aagtccggac tcagatctcg agaggaggag gaggagacag acagcaggat
     2641 gccccacctc gacagccccg gcagctccca gccgagacgc tccttcctct caagggtgat
     2701 cagggcagcg ctaccgttgc agctgcttct gctgctgctg ctgctcctgg cctgcctgct
     2761 acctgcctct gaagatgact acagctgcac ccaggccaac aactttgccc gatccttcta
     2821 ccccatgctg cggtacacca acgggccacc tcccacctag gaattcgata tcaagcttat
     2881 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2941 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3001 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3061 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3121 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3181 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3241 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     3301 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3361 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3421 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     3481 ccgagcgcta ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtcac
     3541 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     3601 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     3661 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     3721 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     3781 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3841 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3901 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3961 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4021 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4081 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     4141 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4201 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4261 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4321 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4381 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4441 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4501 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4561 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4621 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4681 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4741 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4801 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4861 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4921 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4981 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5041 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5101 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5161 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5221 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5281 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5341 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5401 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5461 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5521 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5581 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5641 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5701 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5761 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5821 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5881 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5941 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6001 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6061 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6121 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6181 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6241 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
