LOCUS       sequence-128335-        6038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA to the vesicular GABA transporter VGAT. Cre 
            dependent expression by flex switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6038)
  AUTHORS   Yu X, Ye Z, Houston CM, Zecharia AY, Ma Y, Zhang Z, Uygun DS, Parker
            S, Vyssotski AL, Yustos R, Franks NP, Brickley SG, Wisden W
  TITLE     Wakefulness Is Governed by GABA and Histamine Cotransmission.
  JOURNAL   Neuron. 2015 Jun 17. pii: S0896-6273(15)00516-4. doi: 
            10.1016/j.neuron.2015.06.003.
  PUBMED    26094607
REFERENCE   2  (bases 1 to 6038)
  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: "Neuron. 
            2015 Jun 17. pii: S0896-6273(15)00516-4. doi: 
            10.1016/j.neuron.2015.06.003."
COMMENT     Sequence Label: pAAV-hsyn-flex-dsRed-shvgat
FEATURES             Location/Qualifiers
     source          1..6038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        186..633
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(671..704)
                     /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(755..788)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(955..1081)
                     /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(1109..1123)
                     /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(1151..1272)
                     /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(1322..1999)
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MDSSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(1384..1407)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1582..1601)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1799..1819
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1851..1869
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    2023..2056
                     /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    2107..2140
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2165..2753
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2218..2238)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2636..2647)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2785..3261
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2984..3003)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3301..3441
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3301..3430
                     /label=AAV2 ITR
     rep_origin      3516..3971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3603..3622)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3813..3834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3988..4007)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4107..4129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4167..4185)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4253..4357
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4358..4426,4427..5218)
                     /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(4576..4595)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5389..5977
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5878..5897
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagactgcag agggccctgc
      181 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      241 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      301 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      361 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
      421 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
      481 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
      541 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
      601 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt
      661 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
      721 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
      781 gaagttatgg cgcgccgacc aacgtctcat tttcgccaaa agttggccca gggcttcccg
      841 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
      901 tattcggtcg aacgcgtcgc cgcgtgttta aacgcattag tcttccaatt gaaaaaagtg
      961 atttaattta taccatttta attcagcttt gtaaaaatgt atcaaagaga tagcaaggta
     1021 ttcagtttta gtaaacaaga taattgctcc taaagtagcc ccttgaattc cgaggcagta
     1081 ggcaaactca tcttgtgcaa tgtatctaca tctgtggctt cactagatac attgcacaag
     1141 atgagtgcgc tcactgtcaa cagcaatata ccttctcgag ccttctgttg ggttaacctg
     1201 aagaagtaat cccagcaagt gtttccaaga tgtgcaggca acgattctgt aaagtactga
     1261 agcctcattc aacatagtat atgtgctgcc gaagcgagca cttaacaagg cttgcggccg
     1321 cctacaggaa caggtggtgg cggccctcgg cgcgctcgta ctgctccacg atggtgtagt
     1381 cctcgttgtg ggaggtgatg tccagcttgg agtccacgta gtagtagccg ggcagctgca
     1441 cgggcttctt ggccatgtag atggacttga actccaccag gtagtggccg ccgtccttca
     1501 gcttcagggc cttgtggatc tcgcccttca gcacgccgtc gcgggggtac aggcgctcgg
     1561 tggaggcctc ccagcccata gtcttcttct gcattacggg gccgtcggag gggaagttca
     1621 cgccgatgaa cttcaccttg tagatgaagg agccgtcctg cagggaggag tcctgggtca
     1681 cggtcaccac gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga
     1741 aggacagctt cttgtagtcg gggatgtcgg cggggtgctt cacgtacacc ttggagccgt
     1801 actggaactg gggggacagg atgtcccagg cgaagggcag ggggccgccc ttggtcacct
     1861 tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga
     1921 actcgtggcc gttcacggag ccctccatgc gcaccttgaa gcgcatgaac tccttgatga
     1981 cgtcctcgga ggagtccatc ctgcaggccc ggtagcgcta gcataacttc gtataaagta
     2041 tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg
     2101 caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat
     2161 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2221 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     2281 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     2341 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     2401 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     2461 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     2521 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     2581 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     2641 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     2701 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     2761 ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     2821 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     2881 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     2941 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     3001 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     3061 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     3121 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     3181 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     3241 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     3301 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3361 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3421 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     3481 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3541 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3601 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3661 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3721 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3781 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3841 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     3901 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3961 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4021 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4081 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4141 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4201 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4261 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4321 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4381 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4441 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4501 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4561 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4621 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4681 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4741 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4801 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4861 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4921 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4981 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5041 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5101 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5161 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5221 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5281 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5341 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5401 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5461 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5521 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5581 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5641 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5701 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5761 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5821 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5881 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5941 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6001 acggttcctg gccttttgct ggccttttgc tcacatgt
//
