LOCUS       sequence-355964-        7157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses scramble shRNA for control expts. Flexed cassette driven 
            by pan neuronal gene promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7157)
  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 7157)
  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-flex-GFP-shRNA-scramble
FEATURES             Location/Qualifiers
     source          1..7157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      111..566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(198..217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     408..429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(583..602)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     702..724
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(762..780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        848..952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(953..1021,1022..1813)
                     /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(1171..1190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1984..2572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2473..2492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2634..2763
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(4631..4664)
                     /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(4715..4748)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(4915..5041)
                     /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(5069..5083)
                     /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(5110..5231)
                     /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(5280..5993,5994..5996,5997..5999))
                     /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(5283..5993,5994..5996,5997..5999))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5325..5346)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5674..5693
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5933..5954
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      5996..6005
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6022..6055
                     /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    6106..6139
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6164..6752
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6217..6237)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6635..6646)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6983..7002)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
       61 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
      121 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      181 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      241 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      301 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
      361 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      421 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
      481 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      541 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
      601 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      661 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      721 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
      781 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
      841 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      901 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      961 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1021 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1081 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1141 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1201 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1261 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1321 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1381 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1441 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1501 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1561 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1621 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1681 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1741 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1801 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1861 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1921 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1981 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2041 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2101 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2161 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2221 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2281 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2341 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2401 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2461 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2521 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2581 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     2641 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
     2701 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     2761 cctgcggccg cacgcgtgtg tctagactgc agagggccct gcgtatgagt gcaagtgggt
     2821 tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg
     2881 gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga
     2941 aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg
     3001 cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc
     3061 gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc
     3121 ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc
     3181 tgcggcgccg gcgactcagc gctgatgaat cccctaatga tttttatcaa aatcattaag
     3241 ttaaggtaga tacacatctt gtcatatgat caaatggttt cgccaaaaat caataatcag
     3301 acaacaaaat gtgcgaactc gatattttac acgactctct ttaccaattc tgccccgaat
     3361 tacacttaaa acgactcaac agcttaacgt tggcttgcca cgccttactt gactgtaaaa
     3421 ctctcactct taccgaactt ggccgtaacc tgccaaccaa agcgagaaca aaacataaca
     3481 tcaaacgaat cgaccgattg ttaggtaatc gtcacctcca caaagagcga ctcgctgtat
     3541 accgttggca tgctagcttt atctgttcgg gcaatacgat gcccattgta cttgttgact
     3601 ggtctgatat ccgtgagcaa aaacggctta tggtattgcg agcttcagtc gcactacacg
     3661 gtcgttctgt tactctttat gagaaagcgt tcccgctttc agagcaatgt tcaaagaaag
     3721 ctcatgacca atttctagcc gaccttgcga gcattctacc gagtaacacc acaccgctca
     3781 ttgtcagtga tgctggcttt aaagtgccat ggtataaatc cgttgagaag ctgggttggt
     3841 actggttaag tcgagtaaga ggaaaagtac aatatgcaga cctaggagcg gaaaactgga
     3901 aacctatcag caacttacat gatatgtcat ctagtcactc aaagacttta ggctataaga
     3961 ggctgactaa aagcaatcca atctcatgcc aaattctatt gtataaatct cgctctaaag
     4021 gccgaaaaaa tcagcgctcg acacggactc attgtcacca cccgtcacct aaaatctact
     4081 cagcgtcggc aaaggagcca tggattctag caactaactt acctgttgaa attcgaacac
     4141 ccaaacaact tgttaatatc tattcgaagc gaatgcagat tgaagaaacc ttccgagact
     4201 tgaaaagtcc tgcctacgga ctaggcctac gccatagccg aacgagcagc tcagagcgtt
     4261 ttgatatcat gctgctaatc gccctgatgc ttcaactaac atgttggctt gcgggcgttc
     4321 atgctcagaa acaaggttgg gacaagcact tccaggctaa cacagtcaga aatcgaaacg
     4381 tactctcaac agttcgctta ggcatggaag ttttgcggca ttctggctac acaataacaa
     4441 gggaagactt actcgtggct gcaaccctac tagctcaaaa tttattcaca catggttacg
     4501 ctttggggaa attatgaggg gatctctcag gactcagcgc tgcctcagtc tgcggtgggc
     4561 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt
     4621 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
     4681 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
     4741 gaagttatgg cgcgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg
     4801 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     4861 tattcggtcg aacgcgtcgc cgcgtgttta aacgcattag tcttccaatt gaaaaaagtg
     4921 atttaattta taccatttta attcagcttt gtaaaaatgt atcaaagaga tagcaaggta
     4981 ttcagtttta gtaaacaaga taattgctcc taaagtagcc ccttgaattc cgaggcagta
     5041 ggcaggctcc cgctgaattg gaatcctaca tctgtggctt cactaggatt ccaattcacg
     5101 ggagctcgct cactgtcaac agcaatatac cttctcgagc cttctgttgg gttaacctga
     5161 agaagtaatc ccagcaagtg tttccaagat gtgcaggcaa cgattctgta aagtactgaa
     5221 gcctcattca acatagtata tgtgctgccg aagcgagcac ttaacaaggc ttgcggccgc
     5281 tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     5341 accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag
     5401 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg
     5461 gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg
     5521 ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg
     5581 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     5641 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     5701 aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     5761 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
     5821 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
     5881 taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     5941 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg
     6001 gtggcgaccg gtagcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc
     6061 ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat
     6121 gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac ctctggatta
     6181 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     6241 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     6301 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     6361 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     6421 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     6481 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     6541 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     6601 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     6661 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     6721 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     6781 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     6841 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     6901 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     6961 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     7021 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     7081 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     7141 cggggtttca ccatatt
//
