LOCUS       sequence-489355-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-transgene knocking down adra2a receptor (adrenergic 2a receptor)
            transcripts cell type-selectively. Using the pPRIME. system, this 
            generates mir30-derived shRNAs and a marker from the same RNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  TITLE     Knockdown of adra2a receptors cell type selectively
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5918)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hsynapsin-DIO-adra2a-shRNA-mCherry
FEATURES             Location/Qualifiers
     source          1..5918
                     /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(973..987)
                     /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(1015..1136)
                     /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(1186..1896)
                     /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(1464..1483)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1733..1751
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    1903..1936
                     /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    1987..2020
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2045..2633
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2098..2118)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2516..2527)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2665..3141
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2864..2883)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3181..3321
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3181..3310
                     /label=AAV2 ITR
     rep_origin      3396..3851
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3483..3502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3693..3714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3868..3887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3987..4009
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4047..4065)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4133..4237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4238..4306,4307..5098)
                     /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(4456..4475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5269..5857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5758..5777
                     /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 agtaggcagg caacgtgctg
      961 gttattatcg tacatctgtg gcttcactac gataataacc agcacgttgc tcgctcactg
     1021 tcaacagcaa tataccttct cgagccttct gttgggttaa cctgaagaag taatcccagc
     1081 aagtgtttcc aagatgtgca ggcaacgatt ctgtaaagta ctgaagcctc attcaacata
     1141 gtatatgtgc tgccgaagcg agcacttaac aaggcttgcg agctcttact tgtacagctc
     1201 gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta
     1261 gtcctcgttg tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg
     1321 cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt
     1381 cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc
     1441 ggaggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt
     1501 ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt
     1561 cacggtcacc acgccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg
     1621 gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc
     1681 gtacatgaac tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac
     1741 cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc
     1801 gaactcgtgg ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat
     1861 gatggccatg ttatcctcct cgcccttgct caccatgcta gcataacttc gtataaagta
     1921 tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg
     1981 caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat
     2041 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2101 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     2161 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     2221 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     2281 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     2341 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     2401 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     2461 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     2521 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     2581 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     2641 ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     2701 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     2761 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     2821 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     2881 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     2941 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     3001 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     3061 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     3121 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     3181 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3241 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3301 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     3361 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3421 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     3481 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3541 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3601 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3661 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3721 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     3781 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3841 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3901 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3961 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4021 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4081 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4141 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4201 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4261 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4321 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4381 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4441 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4501 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4561 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4621 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4681 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4741 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4801 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4861 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4921 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4981 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5041 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5101 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5161 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5221 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5281 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5341 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5401 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5461 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5521 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5581 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5641 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5701 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5761 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5821 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5881 acggttcctg gccttttgct ggccttttgc tcacatgt
//
