LOCUS       sequence-158053-        6279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone with a full length H1/TO promoter driving expression of
            a sgRNA that targets exon 3 of the mouse Tet2 locus. 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 6279)
  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 6279)
  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: 
            pJEP13-AAV-H1/TO-L-sgRNA(Tet2)-CMV-TetR-P2A-eGFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..6279
                     /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
     promoter        286..380
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     297..316
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    334..352
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    362..380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        405..480
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        514..817
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        818..1021
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     972..992
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1018..1042
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1166..1789
                     /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(1242..1261)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1808..1861
                     /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(1862..1864,1865..2575)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1862..1864,1865..2575)
                     /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(1904..1925)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2165..2184)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2512..2533
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2867..3455
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2920..2940)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3338..3349)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3542..3682
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3542..3671
                     /label=AAV2 ITR
     rep_origin      3757..4212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3844..3863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4054..4075
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4229..4248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4348..4370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4408..4426)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4494..4598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4599..4667,4668..5459)
                     /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(4817..4836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5630..6218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6119..6138
                     /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 cgcgtgaatt cgaacgctga cgtcatcaac
      181 ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc
      241 ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc
      301 tatgtgttct gggaaatcac cataaacgtg aaatccctat cagtgataga gacttataag
      361 ttccctatca gtgatagaga caccgaaagt gccaacagat atccgtttta gagctagaaa
      421 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      481 ttttttaccg gtccatggac tagttctaga gtccgttaca taacttacgg taaatggccc
      541 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      601 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      661 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      721 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      781 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      841 caatgggcgt gaatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      901 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      961 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1021 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
     1081 aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa cgctgcagga
     1141 attgatccgc ggccgcaccg gtaccatgat gtccagatta gataaaagta aagtgattaa
     1201 cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc
     1261 ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt
     1321 gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt gccctttaga
     1381 aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact
     1441 aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga
     1501 aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt
     1561 atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag atcaagagca
     1621 tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg
     1681 acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga
     1741 attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtccg cggctagcgg
     1801 cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     1861 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1921 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1981 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2041 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2101 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2161 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2221 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2281 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2341 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2401 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2461 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2521 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     2581 actcagatct cgagaggagg aggaggagac agacagcagg atgccccacc tcgacagccc
     2641 cggcagctcc cagccgagac gctccttcct ctcaagggtg atcagggcag cgctaccgtt
     2701 gcagctgctt ctgctgctgc tgctgctcct ggcctgcctg ctacctgcct ctgaagatga
     2761 ctacagctgc acccaggcca acaactttgc ccgatccttc taccccatgc tgcggtacac
     2821 caacgggcca cctcccacct aggaattcga tatcaagctt atcgataatc aacctctgga
     2881 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2941 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3001 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3061 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3121 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3181 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3241 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     3301 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3361 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3421 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc taataaaaga
     3481 tctttatttt cattagatct gtgtgttggt tttttgtgtc acgtgcggac cgagcggccg
     3541 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3601 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3661 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3721 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3781 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3841 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3901 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3961 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4021 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4081 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     4141 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4201 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4261 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4321 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4381 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4441 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4561 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4621 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4681 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4741 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4801 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4861 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4921 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4981 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5041 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5101 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5161 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5221 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5281 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5341 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5401 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5461 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5521 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5581 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5641 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5701 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5761 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5821 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5881 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5941 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6001 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6061 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6121 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6181 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6241 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
