LOCUS       Exported                6272 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Dox-inducible H1 driven SaCas9 gRNA expression cassette without a 
            gRNA. Followed by an EFS driven GFP-KASH in a separate reading 
            frame. SapI can be used to clone in a gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    
                pJEP323-pAAV-FullH1TO-SaCa9gRNAi(SapI)-CMV-TetR-2A-EGFP-K...
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6272)
  AUTHORS   Kumar N, Stanford W, Aradhana CS, Abraham ND, Dao TJ, Thaseen S, 
            Sairavi A, Gonzalez CU, Ploski JE.
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front. Mol. Neurosci. (2018) 13
REFERENCE   2  (bases 1 to 6272)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6272
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..95
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     12..31
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    49..67
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    77..95
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        118..193
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        226..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     730..754
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             884..1501
                     /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(957..976)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1523..1576
                     /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             1577..2290
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1619..1640)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1880..1899)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2227..2248
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2582..3170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2635..2655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3053..3064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3257..3397
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3257..3386
                     /label=AAV2 ITR
     rep_origin      3472..3927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3559..3578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3769..3790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3944..3963)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4063..4085
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4123..4141)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4209..4313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4314..5174
                     /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(4532..4551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5345..5933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5834..5853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5995..6124
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatc cctatcagtg
       61 atagagactt ataagttccc tatcagtgat agagacaccg gaagagcgag ctcttctgtt
      121 ttagtactct ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa
      181 cttgttggcg agatttttac cggtccatgg actagttcta gagtccgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      781 tgacctccat agaagacacc gggaccgatc cagcctccgc ggccgggaac ggtgcattgg
      841 aacggacctg caggaattga tccgcggccg caccggtacc atgatgtcca gattagataa
      901 aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg aaggtttaac
      961 aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt ggcatgtaaa
     1021 aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc accatactca
     1081 cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg ctaaaagttt
     1141 tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta cacggcctac
     1201 agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac aaggtttttc
     1261 actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag gttgcgtatt
     1321 ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta ctgatagtat
     1381 gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag agccagcctt
     1441 cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg
     1501 gtccgcggct agcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga
     1561 ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg tacccatcct
     1621 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1681 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1741 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1801 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1861 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1921 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1981 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2041 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2101 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2161 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2221 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2281 gctgtacaag tccggactca gatctcgaga ggaggaggag gagacagaca gcaggatgcc
     2341 ccacctcgac agccccggca gctcccagcc gagacgctcc ttcctctcaa gggtgatcag
     2401 ggcagcgcta ccgttgcagc tgcttctgct gctgctgctg ctcctggcct gcctgctacc
     2461 tgcctctgaa gatgactaca gctgcaccca ggccaacaac tttgcccgat ccttctaccc
     2521 catgctgcgg tacaccaacg ggccacctcc cacctaggaa ttcgatatca agcttatcga
     2581 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2641 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2701 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2761 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2821 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2881 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2941 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3001 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3061 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3121 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3181 agcgctaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtcacgtg
     3241 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3301 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3361 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3421 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3481 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3541 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3601 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3661 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3721 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3781 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     3841 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3901 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3961 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4021 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4081 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4141 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4201 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4261 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4321 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4381 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4441 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4501 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4561 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4621 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4681 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4741 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4801 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4861 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4921 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4981 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5041 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5101 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5161 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5221 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5281 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5341 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5401 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5461 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5521 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5581 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5641 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5701 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5761 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5821 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5881 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5941 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca
     6001 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc
     6061 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
     6121 tcctgcggcc gcactgacgc gtgaattcga acgctgacgt catcaacccg ctccaaggaa
     6181 tcgcgggccc agtgtcacta ggcgggaaca cccagcgcgc gtgcgccctg gcaggaagat
     6241 ggctgtgagg gacaggggag tggcgccctg ca
//
