LOCUS       Exported                6150 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  AAV backbone with a minimal 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    pJEP15-AAV-H1/TO-sgRNA(Tet2)-CMV-TetR-P2A-eGFP-KASH-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  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 6150)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6150
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        276..351
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        385..688
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        689..892
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     843..863
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     889..913
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1040..1657
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(1113..1132)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1679..1732
                     /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             1733..2446
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1733..2446
                     /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(1775..1796)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2036..2055)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2383..2404
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2738..3326
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2791..2811)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3209..3220)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3413..3553
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3413..3542
                     /label=AAV2 ITR
     rep_origin      3628..4083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3715..3734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3925..3946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4100..4119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4219..4241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4279..4297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4365..4469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4470..5330
                     /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(4688..4707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5501..6089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5990..6009
                     /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 acaccgaaag tgccaacaga tatccgtttt agagctagaa atagcaagtt
      301 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttacc
      361 ggtccatgga ctagttctag agtccgttac ataacttacg gtaaatggcc cgcctggctg
      421 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      481 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      541 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      601 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      661 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      721 tgaatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      781 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      841 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
      901 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
      961 ggaccgatcc agcctccgcg gccgggaacg gtgcattgga acgctgcagg aattgatccg
     1021 cggccgcacc ggtaccatga tgtccagatt agataaaagt aaagtgatta acagcgcatt
     1081 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     1141 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     1201 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     1261 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     1321 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     1381 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     1441 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     1501 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     1561 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     1621 atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc gcggctagcg gcagtggaga
     1681 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa
     1741 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1801 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1861 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1921 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1981 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2041 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2101 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2161 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2221 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2281 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2341 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2401 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg gactcagatc
     2461 tcgagaggag gaggaggaga cagacagcag gatgccccac ctcgacagcc ccggcagctc
     2521 ccagccgaga cgctccttcc tctcaagggt gatcagggca gcgctaccgt tgcagctgct
     2581 tctgctgctg ctgctgctcc tggcctgcct gctacctgcc tctgaagatg actacagctg
     2641 cacccaggcc aacaactttg cccgatcctt ctaccccatg ctgcggtaca ccaacgggcc
     2701 acctcccacc taggaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     2761 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2821 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2881 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2941 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3001 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3061 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3121 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3181 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3241 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3301 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctaataaaag atctttattt
     3361 tcattagatc tgtgtgttgg ttttttgtgt cacgtgcgga ccgagcggcc gcaggaaccc
     3421 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3481 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3541 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3601 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     3661 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3721 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3781 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3841 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3901 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3961 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4021 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4081 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4141 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4201 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4261 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4321 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4381 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4441 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4501 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4561 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4621 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4681 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4741 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4801 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4861 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4921 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4981 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5041 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5101 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5161 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5221 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5281 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5341 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5401 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5461 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5521 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5581 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5641 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5701 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5761 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5821 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5881 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5941 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6001 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6061 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6121 tggccttttg ctggcctttt gctcacatgt
//
