LOCUS       Exported                5683 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pAAV plasmid with BspQI sites for insertion of target specific 
            shRNAs in frame of the murine U6 promoter and co expressing 
            mCherry.3XFLAG under the hSyn promoter .
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-U6-hSyn::mCherry.3xFLAG-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5683)
  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 5683)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5683
                     /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        526..973
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1005..1014
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1011..1718
                     /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(1156..1174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1424..1443
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1725..1790
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1810..2398
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1863..1883)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2281..2292)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2430..2906
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2629..2648)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2946..3086
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2946..3075
                     /label=AAV2 ITR
     rep_origin      3161..3616
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3248..3267)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3458..3479
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3633..3652)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3752..3774
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3812..3830)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3898..4002
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4003..4863
                     /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(4221..4240)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5034..5622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5523..5542
                     /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 tttgtgaaga gcgagctcgc
      481 tcttcgtttt cgcgtgtgtc tagactgcag agggccctgc gtatgagtgc aagtgggttt
      541 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      601 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
      661 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
      721 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
      781 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
      841 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
      901 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
      961 cctgagagcg cagtcgagaa ggtaccggat cctctagagt cgacgccacc atggtgagca
     1021 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     1081 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1141 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1201 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1261 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1321 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1381 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1441 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     1501 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     1561 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     1621 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1681 gccgccactc caccggcggc atggacgagc tgtacaagga attcgactac aaagaccatg
     1741 acggtgatta taaagatcat gatattgatt acaaggatga cgatgacaag ctagcataag
     1801 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     1861 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     1921 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     1981 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2041 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2101 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2161 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     2221 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     2281 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     2341 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     2401 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     2461 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     2521 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     2581 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     2641 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     2701 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     2761 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     2821 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     2881 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     2941 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3001 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3061 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3121 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3181 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3241 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3301 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3361 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3421 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3481 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     3541 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3601 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3661 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3721 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3781 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3841 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3901 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3961 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4021 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4081 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4141 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4201 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4261 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4321 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4381 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4441 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4501 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4561 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4621 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4681 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4741 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4801 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4861 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4921 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4981 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5041 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5101 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5161 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5221 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5281 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5341 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5401 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5461 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5521 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5581 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5641 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
