LOCUS       sequence-489358-        5916 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  AAV transgene designes to express scramble (control) shRNA sequence 
            cell-type selectively. Scramble hairpin expressed from the mir30 
            cassette together with mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  TITLE     Knockdown of adra2a receptors cell type selectively
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5916)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hsynapsin-DIO-scramble shRNA - mCherry
FEATURES             Location/Qualifiers
     source          1..5916
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(819..945)
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(972..986)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(1013..1134)
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             complement(1184..1894)
                     /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(1462..1481)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1731..1749
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    1901..1934
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1985..2018
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2043..2631
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2096..2116)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2514..2525)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2663..3139
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2862..2881)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3179..3319
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3179..3308
                     /label=AAV2 ITR
     rep_origin      3394..3849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3481..3500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3691..3712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3866..3885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3985..4007
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4045..4063)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4131..4235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4236..4304,4305..5096)
                     /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(4454..4473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5267..5855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5756..5775
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gcaggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccgcgtg tttaaacgca ttagtcttcc aattgaaaaa agtgatttaa tttataccat
      841 tttaattcag ctttgtaaaa atgtatcaaa gagatagcaa ggtattcagt tttagtaaac
      901 aagataattg ctcctaaagt agccccttga attccgaggc agtaggcagc cgcgattagg
      961 ctgttataat acatctgtgg cttcactatt ataacagcct aatcgcggcc gctcactgtc
     1021 aacagcaata taccttctcg agccttctgt tgggttaacc tgaagaagta atcccagcaa
     1081 gtgtttccaa gatgtgcagg caacgattct gtaaagtact gaagcctcat tcaacatagt
     1141 atatgtgctg ccgaagcgag cacttaacaa ggcttgcgag ctcttacttg tacagctcgt
     1201 ccatgccgcc ggtggagtgg cggccctcgg cgcgttcgta ctgttccacg atggtgtagt
     1261 cctcgttgtg ggaggtgatg tccaacttga tgttgacgtt gtaggcgccg ggcagctgca
     1321 cgggcttctt ggccttgtag gtggtcttga cctcagcgtc gtagtggccg ccgtccttca
     1381 gcttcagcct ctgcttgatc tcgcccttca gggcgccgtc ctcggggtac atccgctcgg
     1441 aggaggcctc ccagcccatg gtcttcttct gcattacggg gccgtcggag gggaagttgg
     1501 tgccgcgcag cttcaccttg tagatgaact cgccgtcctg cagggaggag tcctgggtca
     1561 cggtcaccac gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga
     1621 aggacagctt caagtagtcg gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt
     1681 acatgaactg aggggacagg atgtcccagg cgaagggcag ggggccaccc ttggtcacct
     1741 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga
     1801 actcgtggcc gttcacggag ccctccatgt gcaccttgaa gcgcatgaac tccttgatga
     1861 tggccatgtt atcctcctcg cccttgctca ccatgctagc ataacttcgt ataaagtatc
     1921 ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     1981 aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg
     2041 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2101 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2161 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     2221 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     2281 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     2341 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     2401 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     2461 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     2521 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     2581 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     2641 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     2701 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     2761 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     2821 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     2881 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     2941 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     3001 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     3061 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     3121 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     3181 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     3241 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     3301 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3361 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     3421 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     3481 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3541 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3601 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3661 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3721 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     3781 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3841 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3901 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3961 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4021 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4081 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4141 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4201 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4261 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4321 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4381 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4441 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4501 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4561 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4621 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4681 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4741 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4801 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4861 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4921 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4981 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5041 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5101 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5161 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5221 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5281 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5341 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5401 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5461 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5521 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5581 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5641 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5701 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5761 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5821 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5881 ggttcctggc cttttgctgg ccttttgctc acatgt
//
