LOCUS       sequence-163793-        6151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone with a minimal H1/TO promoter driving expression of a 
            sgRNA. The gRNA can be inserted via Sapl. 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 6151)
  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 6151)
  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: 
            pJEP14-AAV-H1/TO-sgRNA(Empty)-CMV-TetR-P2A-eGFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..6151
                     /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        157..251
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     168..187
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    205..223
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        274..349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        383..686
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        687..890
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     841..861
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     887..911
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1038..1661
                     /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(1114..1133)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1680..1733
                     /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(1734..1736,1737..2447)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1734..1736,1737..2447)
                     /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(1776..1797)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2037..2056)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2384..2405
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2739..3327
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2792..2812)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3210..3221)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3414..3554
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3414..3543
                     /label=AAV2 ITR
     rep_origin      3629..4084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3716..3735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3926..3947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4101..4120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4220..4242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4280..4298)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4366..4470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4471..4539,4540..5331)
                     /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(4689..4708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5502..6090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5991..6010
                     /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 cgcgtaatat ttgcatgtcg ctatgtgttc
      181 tgggaaatca ccataaacgt gaaatcccta tcagtgatag agacttataa gttccctatc
      241 agtgatagag acaccggaag agcgagctct tctgttttag agctagaaat agcaagttaa
      301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttaccgg
      361 tccatggact agttctagag tccgttacat aacttacggt aaatggcccg cctggctgac
      421 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      481 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      541 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      601 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      661 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      721 aatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      781 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      841 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
      901 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      961 accgatccag cctccgcggc cgggaacggt gcattggaac ggacctgcag gaattgatcc
     1021 gcggccgcac cggtaccatg atgtccagat tagataaaag taaagtgatt aacagcgcat
     1081 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     1141 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg
     1201 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     1261 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     1321 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     1381 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac
     1441 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg
     1501 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     1561 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     1621 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc cgcggctagc ggcagtggag
     1681 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca
     1741 agggcgagga gctgttcacc ggggtggtac ccatcctggt cgagctggac ggcgacgtaa
     1801 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1861 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1921 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1981 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2041 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2101 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2161 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2221 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2281 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2341 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2401 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat
     2461 ctcgagagga ggaggaggag acagacagca ggatgcccca cctcgacagc cccggcagct
     2521 cccagccgag acgctccttc ctctcaaggg tgatcagggc agcgctaccg ttgcagctgc
     2581 ttctgctgct gctgctgctc ctggcctgcc tgctacctgc ctctgaagat gactacagct
     2641 gcacccaggc caacaacttt gcccgatcct tctaccccat gctgcggtac accaacgggc
     2701 cacctcccac ctaggaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     2761 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     2821 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     2881 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     2941 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3001 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3061 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3121 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3181 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3241 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3301 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctaataaaa gatctttatt
     3361 ttcattagat ctgtgtgttg gttttttgtg tcacgtgcgg accgagcggc cgcaggaacc
     3421 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3481 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3541 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3601 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     3661 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3721 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3781 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3841 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3901 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3961 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4021 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4081 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4141 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4201 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4261 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4321 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4441 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4501 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4561 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4621 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4681 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4741 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4801 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4861 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4921 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4981 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5041 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5101 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5161 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5221 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5281 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5341 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5401 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5461 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5521 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5581 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5641 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5701 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5761 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5821 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5881 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5941 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6001 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6061 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6121 ctggcctttt gctggccttt tgctcacatg t
//
