LOCUS Exported 5707 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pAAV plasmid expressing Diras2 shRNA1 under the U6 promoter and mCherry.3XFLAG under the hSyn promoter. ACCESSION . VERSION . KEYWORDS pAAV-U6-Diras2_1-hSyn::mCherry.3xFLAG-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5707) AUTHORS Grunewald L, Chiocchetti AG, Weber H, Scholz CJ, Schartner C, Freudenberg F, Reif A TITLE Knockdown of the ADHD Candidate Gene Diras2 in Murine Hippocampal Primary Cells. JOURNAL J Atten Disord. 2019 Jan 9:1087054718822129. doi: 10.1177/1087054718822129. PUBMED 30623719 REFERENCE 2 (bases 1 to 5707) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5707 /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 150..462 /label=U6 promoter /note="RNA polymerase III promoter for mouse U6 snRNA (Das et al., 1988)" primer_bind 381..401 /label=mU6-F /note="Mouse U6 promoter, forward primer" promoter 550..997 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 1029..1038 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1035..1742 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1180..1198) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1448..1467 /label=mCherry-F /note="mCherry, forward primer" CDS 1749..1814 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" misc_feature 1834..2422 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1887..1907) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2305..2316) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2454..2930 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2653..2672) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 2970..3110 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2970..3099 /label=AAV2 ITR rep_origin 3185..3640 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3272..3291) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3482..3503 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3657..3676) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3776..3798 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3836..3854) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3922..4026 /gene="bla" /label=AmpR promoter CDS 4027..4887 /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(4245..4264) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5058..5646 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5547..5566 /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 gcgttcgaaa tccgacgccg ccatctctag gcccgcgccg 181 gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg ttaatttgca 241 tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga taatctgttc 301 tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag atacaaatac 361 taaattatta ttttaaaaaa cagcacaaaa ggaaactcac cctaactgta aagtaattgt 421 gtgttttgag actataaata tcccttggag aaaagccttg tttgcctcca gatcgacggc 481 aattcaagag attgccgtcg atctggaggt ttttcgcgtg tgtctagact gcagagggcc 541 ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct 601 gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc 661 cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc 721 agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac 781 tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg 841 tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg 901 gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt 961 gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg agaaggtacc ggatcctcta 1021 gagtcgacgc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 1081 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 1141 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 1201 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 1261 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 1321 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 1381 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct 1441 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 1501 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 1561 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 1621 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 1681 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 1741 aggaattcga ctacaaagac catgacggtg attataaaga tcatgatatt gattacaagg 1801 atgacgatga caagctagca taagcttatc gataatcaac ctctggatta caaaatttgt 1861 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 1921 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 1981 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 2041 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 2101 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 2161 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 2221 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc 2281 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 2341 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 2401 tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc 2461 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca 2521 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata 2581 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta 2641 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg 2701 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat 2761 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca 2821 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc 2881 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt 2941 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc 3001 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 3061 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 3121 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 3181 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 3241 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc 3301 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 3361 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 3421 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 3481 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat 3541 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa 3601 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 3661 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 3721 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 3781 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 3841 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 3901 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 3961 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4021 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 4081 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 4141 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 4201 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 4261 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 4321 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 4381 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 4441 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 4501 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4561 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 4621 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4681 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4741 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4801 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4861 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4921 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 4981 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 5041 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 5101 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 5161 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 5221 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 5281 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 5341 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 5401 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 5461 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 5521 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5581 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5641 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 5701 cacatgt //