LOCUS       sequence-365459-        6753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses scramble shRNA for control expts. Flexed (DIO) cassette 
            driven by hdc promoter fragment for pan neuronal expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6753)
  AUTHORS   Miracca G, Anuncibay-Soto B, Tossell K, Yustos R, Vyssotski AL, 
            Franks NP, Wisden W
  TITLE     NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential 
            for Sustaining NREM and REM Sleep.
  JOURNAL   J Neurosci. 2022 May 31. pii: JNEUROSCI.0350-21.2022. doi: 
            10.1523/JNEUROSCI.0350-21.2022.
  PUBMED    35649726
REFERENCE   2  (bases 1 to 6753)
  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 Neurosci.
            2022 May 31. pii: JNEUROSCI.0350-21.2022. doi: 
            10.1523/JNEUROSCI.0350-21.2022."
COMMENT     Sequence Label: pAAV-HDC-DIO-GFP-shRNA.scramble
FEATURES             Location/Qualifiers
     source          1..6753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(1347..1380)
                     /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(1431..1464)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(1631..1757)
                     /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(1785..1799)
                     /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(1826..1947)
                     /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(join(1996..2709,2710..2712,2713..2715))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1999..2709,2710..2712,2713..2715))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2041..2062)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2390..2409
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2649..2670
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2712..2721
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2738..2771
                     /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    2822..2855
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2880..3468
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2933..2953)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3351..3362)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3500..3976
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3699..3718)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4016..4156
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4016..4145
                     /label=AAV2 ITR
     rep_origin      4231..4686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4318..4337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4528..4549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4703..4722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4822..4844
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4882..4900)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4968..5072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5073..5141,5142..5933)
                     /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(5291..5310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6104..6692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6593..6612
                     /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 gcggccgcca tatgaccacc ccccatgaag tctgttgtgt aagtacaggt
      181 aatgctttag acactgtttt tctcagattg tgttcaagct cttgggtgca ggcatccgca
      241 gtccagagcc tctgaggaac aaagggacct cgatataaag aacaaaaagc tctcggtatt
      301 cgggaaaact ccgaaggatt tttgttgttt tgtggcagtg ttggggttca ggacaaacac
      361 accaccccag aatatgattt caggagaaac tgccatcccc gcccccacta gtacgcttcc
      421 tttgtgttat ctcgagaagc cctggacgta ggaggagttc tcaaaagctg ttctttcgta
      481 agagatggtc aggaagaaac gttgattagg aaaagtactc gactcaggaa gaggaggggt
      541 acagataact gctgtcacca gaaagatttc catctgacaa agtggcaatt cttccccctt
      601 acgaatgtgt gtcccgcact ctgctgcacc caggctccgc tctgttctcc agctctgctt
      661 gctgtagaag cgtccatcca ctaaagggac ttccagattt attggcactg aacaggtttt
      721 tcctccgtta gcttaaacag tagtcgaggc aaagaaccca gaacaagtgc tatgtagtaa
      781 tggataaaaa gaatccttca tatcctgttg ttgatctgtc ctgagctcag cctggctagc
      841 tatgggcttc tctgagccgg ctctgccctc acctcataga gaagccaggg caggagcaac
      901 tccaccctct gtgctactta ttgtgaatgc ccttggccta tttctggcct ctgagctagg
      961 agctttattc aaggctgggg ctctatggtc tgaatgcagg ggcagggatg atagagggct
     1021 cttttacctt gtctatccag agctgcttgt aggcttggga ggctaggtgc cccaactggt
     1081 ctaaaaacac catgttctct gattctgttg atcaaagttc aaatcagaaa ggacggggac
     1141 aagagaggct gaaattaagc ctgaaggaag ggactttatg ggggcggggc taaaggaggg
     1201 gaatagggaa ctccttaaat aagagatgca ctggctgcca gagagtgcac agcacagaca
     1261 aaggcgacca agaagccctg ttgacagctg ccttccagcc tcctctgtct gtctgccagg
     1321 aggagcaatc caaggtcgac tccggaataa cttcgtatag gatactttat acgaagttat
     1381 gcagaatggt agctggattg tagctgctat tagcaatatg aaacctctta ataacttcgt
     1441 atagcataca ttatacgaag ttatggcgcg ccgatcaacg tctcattttc gccaaaagtt
     1501 ggcccagggc ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc
     1561 ttccgtcaca ggtatttatt cggtcgaacg cgtcgccgcg tgtttaaacg cattagtctt
     1621 ccaattgaaa aaagtgattt aatttatacc attttaattc agctttgtaa aaatgtatca
     1681 aagagatagc aaggtattca gttttagtaa acaagataat tgctcctaaa gtagcccctt
     1741 gaattccgag gcagtaggca ggctcccgct gaattggaat cctacatctg tggcttcact
     1801 aggattccaa ttcacgggag ctcgctcact gtcaacagca atataccttc tcgagccttc
     1861 tgttgggtta acctgaagaa gtaatcccag caagtgtttc caagatgtgc aggcaacgat
     1921 tctgtaaagt actgaagcct cattcaacat agtatatgtg ctgccgaagc gagcacttaa
     1981 caaggcttgc ggccgctact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     2041 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     2101 ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     2161 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     2221 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     2281 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     2341 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     2401 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     2461 gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt
     2521 cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     2581 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     2641 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2701 gcccttgctc accatggtgg cgaccggtag cgctagcata acttcgtata aagtatccta
     2761 tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag
     2821 aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag cttatcgata
     2881 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2941 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3001 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3061 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3121 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3181 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3241 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3301 cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3361 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3421 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag
     3481 cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
     3541 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
     3601 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa
     3661 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag
     3721 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct
     3781 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt
     3841 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
     3901 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc
     3961 tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa
     4021 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4081 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4141 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4201 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4261 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     4321 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4381 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4441 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4501 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4561 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     4621 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     4681 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     4741 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4801 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4861 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4921 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4981 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5041 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5101 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5161 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5221 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5281 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5341 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5401 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5461 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5521 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5581 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5641 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5701 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5761 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5821 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5881 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5941 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6001 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6061 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6121 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6181 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagyaga gcgcagatac
     6241 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6301 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6361 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6421 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6481 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6541 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6601 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6661 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6721 tcctggcctt ttgctggcct tttgctcaca tgt
//
