LOCUS Exported 6935 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV vector that expresses guide RNAs targeting rat Rosa26 and expresses EGFP reporter. ACCESSION . VERSION . KEYWORDS pOTTC1270 - pAAV Rosa26 gRNA A+B EF1a EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6935) AUTHORS Back S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope BT, Hamra FK, Richie CT, Harvey BK TITLE Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats. JOURNAL Neuron. 2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 10.1016/j.neuron.2019.01.035. PUBMED 30792150 REFERENCE 2 (bases 1 to 6935) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6935 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" misc_RNA complement(183..258) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(281..594) /label=U6 promoter /note="RNA polymerase III promoter for mouse U6 snRNA (Das et al., 1988)" primer_bind complement(342..362) /label=mU6-F /note="Mouse U6 promoter, forward primer" promoter 722..1900 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 953..1891 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1848..1868 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1920..1929 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1926..2645 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1926..2642 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1971..1992) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2232..2251) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2579..2600 /label=EGFP-C /note="EGFP, forward primer" misc_feature 2679..3267 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2732..2752) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3150..3161) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3299..3775 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3498..3517) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" promoter 3817..4057 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 3817..3837 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3988..4007 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4086..4161 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" repeat_region 4198..4338 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4198..4327 /label=AAV2 ITR rep_origin 4413..4868 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4500..4519) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4710..4731 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4885..4904) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5004..5026 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5064..5082) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5150..5254 /gene="bla" /label=AmpR promoter CDS 5255..6115 /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(5473..5492) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6286..6874 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6775..6794 /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 gcgtaagctg gagctccacc gcggcgtctc agtacaaaaa 181 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc 241 tatttctagc tctaaaaccc tatcttccag aaagactcca aacaaggctt ttctccaagg 301 gatatttata gtctcaaaac acacaattac tttacagtta gggtgagttt ccttttgtgc 361 tgttttttaa aataataatt tagtatttgt atctcttata gaaatccaag cctatcatgt 421 aaaatgtagc tagtattaaa aagaacagat tatctgtctt ttatcgcaca ttaagcctct 481 atagttacta ggaaatatta tatgcaaatt aaccggggca ggggagtagc cgagcttctc 541 ccacaagtct gtgcgagggg gccggcgcgg gcctagagat ggcggcgtcg gatctatctg 601 agacgggtac ccaattcgcc ctatagtgag cgcgtaagct ttgcaaagat ggataaagtt 661 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat 721 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 781 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 841 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 901 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 961 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat 1021 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg 1081 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg 1141 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc 1201 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg 1261 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc 1321 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg 1381 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc 1441 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc 1501 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag 1561 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc 1621 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga 1681 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat 1741 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta 1801 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac 1861 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggtaccggat ccaccggtcg 1921 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 1981 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 2041 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 2101 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 2161 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 2221 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 2281 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 2341 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 2401 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 2461 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 2521 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 2581 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 2641 agtaaagcgg ccgcactcga gatatcaagc ttatcgataa tcaacctctg gattacaaaa 2701 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2761 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2821 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2881 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2941 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 3001 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 3061 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc 3121 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 3181 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 3241 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg 3301 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt 3361 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt 3421 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac 3481 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct 3541 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt 3601 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg 3661 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg 3721 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat 3781 tttgtaggta accacgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct 3841 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta 3901 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 3961 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 4021 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcaga tcacgaggga 4081 agaaggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 4141 aaagtggcac cgagtcggtg cttttttgtt ttagagctag tcggaccgag cggccgcagg 4201 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 4261 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 4321 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 4381 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc 4441 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc 4501 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 4561 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 4621 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 4681 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 4741 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg 4801 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 4861 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 4921 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 4981 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 5041 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 5101 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 5161 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 5221 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 5281 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 5341 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 5401 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 5461 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 5521 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 5581 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 5641 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 5701 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 5761 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 5821 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 5881 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 5941 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 6001 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 6061 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 6121 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 6181 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 6241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 6301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 6361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 6421 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 6481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 6541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 6601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 6661 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 6721 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 6781 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 6841 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 6901 gttcctggcc ttttgctggc cttttgctca catgt //