LOCUS       sequence-158051-        6277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone with a full length H1/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 6277)
  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 6277)
  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: 
            pJEP12-AAV-H1/TO-L-sgRNA(Empty)-CMV-TetR-P2A-eGFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..6277
                     /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        403..478
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        512..815
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        816..1019
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     970..990
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1016..1040
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1164..1787
                     /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(1240..1259)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1806..1859
                     /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(1860..1862,1863..2573)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1860..1862,1863..2573)
                     /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(1902..1923)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2163..2182)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2510..2531
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2865..3453
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2918..2938)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3336..3347)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3540..3680
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3540..3669
                     /label=AAV2 ITR
     rep_origin      3755..4210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3842..3861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4052..4073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4227..4246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4346..4368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4406..4424)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4492..4596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4597..4665,4666..5457)
                     /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(4815..4834)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5628..6216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6117..6136
                     /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 caccggaaga gcgagctctt ctgttttaga gctagaaata
      421 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      481 ttttaccggt ccatggacta gttctagagt ccgttacata acttacggta aatggcccgc
      541 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      601 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      661 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      721 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      781 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      841 atgggcgtga atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      901 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      961 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     1021 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     1081 gacaccggga ccgatccagc ctccgcggcc gggaacggtg cattggaacg ctgcaggaat
     1141 tgatccgcgg ccgcaccggt accatgatgt ccagattaga taaaagtaaa gtgattaaca
     1201 gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc
     1261 agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc
     1321 tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag
     1381 gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa
     1441 gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa
     1501 ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat
     1561 atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc
     1621 aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac
     1681 aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat
     1741 tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtccgcg gctagcggca
     1801 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     1861 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1921 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1981 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2041 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2101 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2161 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2221 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2281 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2341 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2401 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2461 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2521 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     2581 tcagatctcg agaggaggag gaggagacag acagcaggat gccccacctc gacagccccg
     2641 gcagctccca gccgagacgc tccttcctct caagggtgat cagggcagcg ctaccgttgc
     2701 agctgcttct gctgctgctg ctgctcctgg cctgcctgct acctgcctct gaagatgact
     2761 acagctgcac ccaggccaac aactttgccc gatccttcta ccccatgctg cggtacacca
     2821 acgggccacc tcccacctag gaattcgata tcaagcttat cgataatcaa cctctggatt
     2881 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2941 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3001 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3061 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3121 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3181 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3241 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3301 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3361 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3421 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgcta ataaaagatc
     3481 tttattttca ttagatctgt gtgttggttt tttgtgtcac gtgcggaccg agcggccgca
     3541 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     3601 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     3661 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3721 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     3781 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3841 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3901 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3961 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4021 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4081 ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4141 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4201 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4261 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4321 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4381 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4441 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4501 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4561 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4621 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4681 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4741 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4801 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4861 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4921 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4981 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5041 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5101 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5161 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5221 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5281 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5341 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5401 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5461 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5521 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5761 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6241 cggttcctgg ccttttgctg gccttttgct cacatgt
//
