LOCUS Exported 3814 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION U6 driven SaCas9 gRNA expression cassette without a gRNA. SapI can be used to clone in gRNAs.. ACCESSION . VERSION . KEYWORDS pJEP316-pAAV-U6SaCas9gRNA(SapI)-pA Empty Cassette SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3814) 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 3814) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3814 /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 145..385 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 145..165 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 316..335 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 412..487 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" polyA_signal 561..1037 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(760..779) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 1077..1217 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 1077..1206 /label=AAV2 ITR rep_origin 1292..1747 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1379..1398) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1589..1610 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(1764..1783) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1883..1905 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1943..1961) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2029..2133 /gene="bla" /label=AmpR promoter CDS 2134..2994 /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(2352..2371) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3165..3753 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3654..3673 /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 gcgtgagggc ctatttccca tgattccttc atatttgcat 181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata 241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa 301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg 361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagta 421 ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt 481 ggcgagattt ttaccggtcc atggactagt tctagtcgat atcaagctta tcgataccga 541 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg 601 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca 661 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca 721 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga 781 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc 841 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt 901 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat 961 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg 1021 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga 1081 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 1141 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 1201 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 1261 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 1321 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct 1381 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 1441 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 1501 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 1561 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 1621 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg 1681 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 1741 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 1801 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 1861 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 1921 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 1981 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2101 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 2221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 2281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 2341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 2401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 2461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 2521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 2581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 2641 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 2701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 2761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 2821 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 2881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 2941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3301 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 3781 ttcctggcct tttgctggcc ttttgctcac atgt //