LOCUS Exported 6274 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Dox-inducible H1 driven SaCas9 gRNA expression cassette with a gRNA targeting CREB. Followed by an EFS driven GFP-KASH in a separate reading frame.. ACCESSION . VERSION . KEYWORDS pJEP325-pAAV-FullH1TO-SaCa9sgRNAi(CREB)-CMV-TetR-2A-EGFP-... SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6274) 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 6274) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6274 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 279..373 /label=H1-2O2 promoter /note="doxycycline-inducible variant of the human H1 RNA promoter (Henriksen et al., 2007)" primer_bind 290..309 /label=H1 /note="Human H1 promoter, forward primer" protein_bind 327..345 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 355..373 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" misc_RNA 398..473 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" enhancer 506..809 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 810..1013 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 964..984 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1010..1034 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1164..1781 /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(1237..1256) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" CDS 1803..1856 /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 1857..2570 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1899..1920) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2160..2179) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2507..2528 /label=EGFP-C /note="EGFP, forward primer" misc_feature 2862..3450 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2915..2935) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3333..3344) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" repeat_region 3537..3677 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3537..3666 /label=AAV2 ITR rep_origin 3752..4207 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3839..3858) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4049..4070 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4224..4243) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4343..4365 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4403..4421) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4489..4593 /gene="bla" /label=AmpR promoter CDS 4594..5454 /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(4812..4831) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5625..6213 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6114..6133 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac tgacgcgtga attcgaacgc tgacgtcatc aacccgctcc 181 aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag 241 gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt 301 tctgggaaat caccataaac gtgaaatccc tatcagtgat agagacttat aagttcccta 361 tcagtgatag agacaccgga gcagacaacc agcagaggtt ttagtactct ggaaacagaa 421 tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agatttttac 481 cggtccatgg actagttcta gagtccgtta cataacttac ggtaaatggc ccgcctggct 541 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 601 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 661 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 721 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 781 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 841 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 901 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 961 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 1021 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 1081 gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacggacctg caggaattga 1141 tccgcggccg caccggtacc atgatgtcca gattagataa aagtaaagtg attaacagcg 1201 cattagagct gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga 1261 agctaggtgt agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg 1321 acgccttagc cattgagatg ttagataggc accatactca cttttgccct ttagaagggg 1381 aaagctggca agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc 1441 atcgcgatgg agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc 1501 tcgaaaatca attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg 1561 cactcagcgc tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag 1621 tcgctaaaga agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag 1681 ctatcgaatt atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga 1741 tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg gtccgcggct agcggcagtg 1801 gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga 1861 gcaagggcga ggagctgttc accggggtgg tacccatcct ggtcgagctg gacggcgacg 1921 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 1981 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 2041 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 2101 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 2161 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 2221 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 2281 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 2341 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 2401 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 2461 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 2521 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggactca 2581 gatctcgaga ggaggaggag gagacagaca gcaggatgcc ccacctcgac agccccggca 2641 gctcccagcc gagacgctcc ttcctctcaa gggtgatcag ggcagcgcta ccgttgcagc 2701 tgcttctgct gctgctgctg ctcctggcct gcctgctacc tgcctctgaa gatgactaca 2761 gctgcaccca ggccaacaac tttgcccgat ccttctaccc catgctgcgg tacaccaacg 2821 ggccacctcc cacctaggaa ttcgatatca agcttatcga taatcaacct ctggattaca 2881 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 2941 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 3001 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac 3061 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 3121 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca 3181 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 3241 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga 3301 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 3361 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 3421 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctaata aaagatcttt 3481 attttcatta gatctgtgtg ttggtttttt gtgtcacgtg cggaccgagc ggccgcagga 3541 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 3601 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 3661 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3721 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 3781 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct 3841 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 3901 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 3961 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 4021 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 4081 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg 4141 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 4201 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 4261 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 4321 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 4381 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 4441 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 4501 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4561 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4621 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4681 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4741 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4801 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 4861 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 4921 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 4981 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 5041 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 5101 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 5161 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 5221 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5281 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5341 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5401 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5461 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5521 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5581 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5641 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5701 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5761 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5821 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5881 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5941 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 6001 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 6061 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 6121 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 6181 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6241 ttcctggcct tttgctggcc ttttgctcac atgt //