LOCUS       sequence-158046-        6298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone with a full length U6/TO promoter driving expression of
            a sgRNA. The gRNA can be inserted via Sapl sites. TracrRNA 
            compatible with SpCas9. gRNA transcription is doxycycline 
            dependent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  AUTHORS   de Solis CA, Ho A, Holehonnur R, Ploski JE
  TITLE     The Development of a Viral Mediated CRISPR/Cas9 System with 
            Doxycycline Dependent gRNA Expression for Inducible In vitro and In 
            vivo Genome Editing.
  JOURNAL   Front Mol Neurosci. 2016 Aug 18;9:70. doi: 10.3389/fnmol.2016.00070.
            eCollection 2016.
  PUBMED    27587996
REFERENCE   2  (bases 1 to 6298)
  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: "2016-08-18"; volume: "9:70. doi"; pages: " 
            10.3389/fnmol.2016.00070. eCollection 2016"
COMMENT     Sequence Label: 
            pJEP10-AAV-U6/TO-gRNA(Empty)-CMV-TetR-P2A-eGFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..6298
                     /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=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        533..836
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        837..1040
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     991..1011
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1037..1061
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1185..1808
                     /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(1261..1280)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1827..1880
                     /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(1881..1883,1884..2594)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1881..1883,1884..2594)
                     /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(1923..1944)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2184..2203)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2531..2552
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2886..3474
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2939..2959)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3357..3368)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3561..3701
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3561..3690
                     /label=AAV2 ITR
     rep_origin      3776..4231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3863..3882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4073..4094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4248..4267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4367..4389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4427..4445)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4513..4617
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4618..4686,4687..5478)
                     /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(4836..4855)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5649..6237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6138..6157
                     /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 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      481 agtggcaccg agtcggtgct tttttaccgg tccatggact agttctagag tccgttacat
      541 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      601 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      661 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      721 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      781 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      841 tgcggttttg gcagtacatc aatgggcgtg aatagcggtt tgactcacgg ggatttccaa
      901 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      961 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1021 aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     1081 gctgttttga cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt
     1141 gcattggaac gctgcaggaa ttgatccgcg gccgcaccgg taccatgatg tccagattag
     1201 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     1261 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     1321 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     1381 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     1441 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     1501 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     1561 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     1621 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     1681 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     1741 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa
     1801 gtgggtccgc ggctagcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
     1861 tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1921 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1981 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2041 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2101 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2161 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2221 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2281 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2341 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2401 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2461 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2521 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2581 acgagctgta caagtccgga ctcagatctc gagaggagga ggaggagaca gacagcagga
     2641 tgccccacct cgacagcccc ggcagctccc agccgagacg ctccttcctc tcaagggtga
     2701 tcagggcagc gctaccgttg cagctgcttc tgctgctgct gctgctcctg gcctgcctgc
     2761 tacctgcctc tgaagatgac tacagctgca cccaggccaa caactttgcc cgatccttct
     2821 accccatgct gcggtacacc aacgggccac ctcccaccta ggaattcgat atcaagctta
     2881 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2941 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3001 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3061 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3121 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3181 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3241 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     3301 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3361 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3421 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     3481 accgagcgct aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtca
     3541 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     3601 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3661 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     3721 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3781 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3841 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3901 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3961 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4021 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4081 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     4141 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4201 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4261 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4321 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4381 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4441 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4501 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4561 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4621 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4681 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4741 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4801 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4861 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4921 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4981 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5041 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5101 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5161 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5221 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5281 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5341 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5401 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5461 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5521 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5581 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5641 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5701 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5761 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     5821 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5881 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5941 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6001 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6061 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6121 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6181 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6241 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
