LOCUS Exported 6297 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 6297) 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 6297) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6297 /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 primer_bind 156..176 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 327..346 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 357..375 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 385..403 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" misc_RNA 424..499 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" enhancer 532..835 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 836..1039 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 990..1010 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1036..1060 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1187..1804 /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(1260..1279) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" CDS 1826..1879 /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 1880..2593 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 1880..2593 /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(1922..1943) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2183..2202) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2530..2551 /label=EGFP-C /note="EGFP, forward primer" misc_feature 2885..3473 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2938..2958) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3356..3367) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" repeat_region 3560..3700 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3560..3689 /label=AAV2 ITR rep_origin 3775..4230 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3862..3881) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4072..4093 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4247..4266) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4366..4388 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4426..4444) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4512..4616 /gene="bla" /label=AmpR promoter CDS 4617..5477 /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(4835..4854) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5648..6236 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6137..6156 /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 cgcgtgaggg cctatttccc atgattcctt 181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaactccc 361 tatcagtgat agagattata tatctcccta tcagtgatag agaccggaag agcgagctct 421 tctgttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gtgtttatct 481 cgtcaacttg ttggcgagat ttttaccggt ccatggacta gttctagagt ccgttacata 541 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 601 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 661 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 721 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 781 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 841 gcggttttgg cagtacatca atgggcgtga atagcggttt gactcacggg gatttccaag 901 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 961 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 1021 ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg 1081 ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc gggaacggtg 1141 cattggaacg ctgcaggaat tgatccgcgg ccgcaccggt accatgatgt ccagattaga 1201 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt 1261 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt 1321 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac 1381 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag 1441 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc 1501 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt 1561 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt 1621 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag 1681 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc 1741 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag 1801 tgggtccgcg gctagcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt 1861 cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 1921 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 1981 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 2041 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 2101 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 2161 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 2221 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 2281 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 2341 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg 2401 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 2461 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa 2521 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 2581 cgagctgtac aagtccggac tcagatctcg agaggaggag gaggagacag acagcaggat 2641 gccccacctc gacagccccg gcagctccca gccgagacgc tccttcctct caagggtgat 2701 cagggcagcg ctaccgttgc agctgcttct gctgctgctg ctgctcctgg cctgcctgct 2761 acctgcctct gaagatgact acagctgcac ccaggccaac aactttgccc gatccttcta 2821 ccccatgctg cggtacacca acgggccacc tcccacctag gaattcgata tcaagcttat 2881 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 2941 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 3001 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 3061 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 3121 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 3181 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 3241 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 3301 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 3361 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 3421 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata 3481 ccgagcgcta ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtcac 3541 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc 3601 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 3661 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt 3721 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc 3781 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 3841 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 3901 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 3961 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc 4021 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4081 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga 4141 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4201 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg 4261 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4321 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4381 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 4441 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 4501 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 4561 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 4621 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 4681 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 4741 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 4801 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 4861 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 4921 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 4981 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 5041 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 5101 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 5161 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 5221 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 5281 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 5341 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 5401 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 5461 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 5521 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 5581 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 5641 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 5701 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 5761 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 5821 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 5881 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 5941 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 6001 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 6061 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 6121 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 6181 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 6241 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //