LOCUS Exported 6971 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION An AAV vector that expresses guide RNAs targeting rat TH and expresses EGFP reporter. ACCESSION . VERSION . KEYWORDS pOTTC1088 - pAAV TH gRNA A+B pair EF1a EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6971) 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 6971) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6971 /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 3858..4098 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 3858..3878 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4029..4048 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4128..4203 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" repeat_region 4234..4374 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4234..4363 /label=AAV2 ITR rep_origin 4449..4904 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4536..4555) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4746..4767 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4921..4940) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5040..5062 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5100..5118) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5186..5290 /gene="bla" /label=AmpR promoter CDS 5291..6151 /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(5509..5528) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6322..6910 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6811..6830 /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 tctaaaacaa gggcttcaga agggccgtca 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 accacctcac tatagggcga attgggtacc cgtctcacag gcggatcgat 3841 ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat 3901 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac 3961 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt 4021 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat 4081 atatcttgtg gaaaggacga aacaccgagg ccgaggctgt cacggtggtt ttagagctag 4141 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 4201 tgcttttttt ctccgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca 4261 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4321 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc 4381 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca 4441 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 4501 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt 4561 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4621 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 4681 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4741 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt 4801 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca 4861 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag 4921 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 4981 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 5041 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 5101 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 5161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 5221 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 5281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 5341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 5401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 5461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 5581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 5641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 5701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 5761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5821 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 5941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 6001 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 6061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 6121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 6181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 6241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 6301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 6361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 6421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 6481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6541 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6601 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6661 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6721 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6781 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6841 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6901 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6961 tgctcacatg t //