LOCUS       sequence-232033-        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible U6 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 6344)
  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 6344)
  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: 
            pJEP321-pAAV-FullU6TO-SaCas9gRNAi(SapI)-CMV-TetR-P2A-GFP-KASH-WPRE-s
            hortPA Empty Cassette
FEATURES             Location/Qualifiers
     source          1..6344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        30..105
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        187..490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..694
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     645..665
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     691..715
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             842..1465
                     /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(918..937)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1484..1537
                     /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(1538..1540,1541..2251)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1538..1540,1541..2251)
                     /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(1580..1601)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1841..1860)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2188..2209
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2543..3131
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2596..2616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3014..3025)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3218..3358
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3218..3347
                     /label=AAV2 ITR
     rep_origin      3433..3888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3905..3924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4024..4046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4084..4102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4170..4274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4275..4343,4344..5135)
                     /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(4493..4512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5306..5894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5795..5814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5956..6085
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     6106..6126
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6277..6296
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    6307..6325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    join(6335..6344,1..9)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 tgatagagac cggaagagcg agctcttctg ttttagtact ctggaaacag aatctactaa
       61 aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt accggtccat
      121 ggactagttc tagggatcga cgagagcagc gcgactttta ccggtccatg gactagttct
      181 agagtccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      241 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      301 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      361 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      421 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      481 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      541 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      601 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      661 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
      721 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
      781 cggccgggaa cggtgcattg gaacggacct gcaggaattg atccgcggcc gcaccggtac
      841 catgatgtcc agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga
      901 ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc
      961 tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat
     1021 gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt
     1081 acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt
     1141 acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt
     1201 tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca
     1261 ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga
     1321 aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca
     1381 ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa
     1441 acaacttaaa tgtgaaagtg ggtccgcggc tagcggcagt ggagagggca gaggaagtct
     1501 gctaacatgc ggtgacgtcg aggagaatcc tggcccagtg agcaagggcg aggagctgtt
     1561 caccggggtg gtacccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1621 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1681 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1741 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1801 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1861 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1921 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1981 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2041 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2101 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2161 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2221 gatcactctc ggcatggacg agctgtacaa gtccggactc agatctcgag aggaggagga
     2281 ggagacagac agcaggatgc cccacctcga cagccccggc agctcccagc cgagacgctc
     2341 cttcctctca agggtgatca gggcagcgct accgttgcag ctgcttctgc tgctgctgct
     2401 gctcctggcc tgcctgctac ctgcctctga agatgactac agctgcaccc aggccaacaa
     2461 ctttgcccga tccttctacc ccatgctgcg gtacaccaac gggccacctc ccacctagga
     2521 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     2581 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     2641 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     2701 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     2761 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     2821 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     2881 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     2941 atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt
     3001 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3061 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3121 cgcctccccg catcgatacc gagcgctaat aaaagatctt tattttcatt agatctgtgt
     3181 gttggttttt tgtgtcacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3241 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3301 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3361 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3421 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     3781 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     3841 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3901 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4081 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4141 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4201 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4261 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4321 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4381 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4441 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4501 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4561 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4621 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4681 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4741 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4801 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4861 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4921 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4981 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5041 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5101 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5161 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5221 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5281 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5341 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5401 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5461 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5521 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5581 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5641 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5701 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5761 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5821 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5881 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5941 cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
     6001 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     6061 gccaactcca tcactagggg ttcctgcggc cgcacgcgta cgcgtgaggg cctatttccc
     6121 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     6181 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     6241 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     6301 tgaaactccc tatcagtgat agagattata tatctcccta tcag
//
