LOCUS       sequence-235541-        5707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV plasmid expressing Diras2 shRNA2 under the U6 promoter and 
            mCherry.3XFLAG under the hSyn promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Atten 
            Disord. 2019 Jan 9:1087054718822129. doi: 10.1177/1087054718822129."
COMMENT     Sequence Label: pAAV-U6-Diras2_2-hSyn::mCherry.3xFLAG-WPRE
FEATURES             Location/Qualifiers
     source          1..5707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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             join(4027..4095,4096..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 tttgcggctg tccatctcca
      481 aattcaagag atttggagat ggacagccgt 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
//
