LOCUS       sequence-355962-        6754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA against mouse NMDA receptor NR1 subunit. Flexed 
            cassette driven by hdc (pan neuronal) gene promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6754)
  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 6754)
  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-GluN1
FEATURES             Location/Qualifiers
     source          1..6754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   64..193
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(1410..1443)
                     /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(1494..1527)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(1694..1820)
                     /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(1848..1862)
                     /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(1890..2011)
                     /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(2060..2773,2774..2776,2777..2779))
                     /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(2063..2773,2774..2776,2777..2779))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2105..2126)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2454..2473
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2713..2734
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2776..2785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2802..2835
                     /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    2886..2919
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2944..3532
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2997..3017)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3415..3426)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3564..4040
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3763..3782)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4080..4220
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4080..4209
                     /label=AAV2 ITR
     rep_origin      4295..4750
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4382..4401)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4592..4613
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4767..4786)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4886..4908
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4946..4964)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5032..5136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5137..5205,5206..5997)
                     /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(5355..5374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6168..6754,1..2)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6657..6676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
       61 tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc
      121 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      181 actaggggtt cctgcggccg ccatatgacc accccccatg aagtctgttg tgtaagtaca
      241 ggtaatgctt tagacactgt ttttctcaga ttgtgttcaa gctcttgggt gcaggcatcc
      301 gcagtccaga gcctctgagg aacaaaggga cctcgatata aagaacaaaa agctctcggt
      361 attcgggaaa actccgaagg atttttgttg ttttgtggca gtgttggggt tcaggacaaa
      421 cacaccaccc cagaatatga tttcaggaga aactgccatc cccgccccca ctagtacgct
      481 tcctttgtgt tatctcgaga agccctggac gtaggaggag ttctcaaaag ctgttctttc
      541 gtaagagatg gtcaggaaga aacgttgatt aggaaaagta ctcgactcag gaagaggagg
      601 ggtacagata actgctgtca ccagaaagat ttccatctga caaagtggca attcttcccc
      661 cttacgaatg tgtgtcccgc actctgctgc acccaggctc cgctctgttc tccagctctg
      721 cttgctgtag aagcgtccat ccactaaagg gacttccaga tttattggca ctgaacaggt
      781 ttttcctccg ttagcttaaa cagtagtcga ggcaaagaac ccagaacaag tgctatgtag
      841 taatggataa aaagaatcct tcatatcctg ttgttgatct gtcctgagct cagcctggct
      901 agctatgggc ttctctgagc cggctctgcc ctcacctcat agagaagcca gggcaggagc
      961 aactccaccc tctgtgctac ttattgtgaa tgcccttggc ctatttctgg cctctgagct
     1021 aggagcttta ttcaaggctg gggctctatg gtctgaatgc aggggcaggg atgatagagg
     1081 gctcttttac cttgtctatc cagagctgct tgtaggcttg ggaggctagg tgccccaact
     1141 ggtctaaaaa caccatgttc tctgattctg ttgatcaaag ttcaaatcag aaaggacggg
     1201 gacaagagag gctgaaatta agcctgaagg aagggacttt atgggggcgg ggctaaagga
     1261 ggggaatagg gaactcctta aataagagat gcactggctg ccagagagtg cacagcacag
     1321 acaaaggcga ccaagaagcc ctgttgacag ctgccttcca gcctcctctg tctgtctgcc
     1381 aggaggagca atccaaggtc gactccggaa taacttcgta taggatactt tatacgaagt
     1441 tatgcagaat ggtagctgga ttgtagctgc tattagcaat atgaaacctc ttaataactt
     1501 cgtatagcat acattatacg aagttatggc gcgccgatca acgtctcatt ttcgccaaaa
     1561 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
     1621 atcttccgtc acaggtattt attcggtcga acgcgtcgcc gcgtgtttaa acgcattagt
     1681 cttccaattg aaaaaagtga tttaatttat accattttaa ttcagctttg taaaaatgta
     1741 tcaaagagat agcaaggtat tcagttttag taaacaagat aattgctcct aaagtagccc
     1801 cttgaattcc gaggcagtag gcatggagtt tgaggcttca cagaatacat ctgtggcttc
     1861 actattctgt gaagcctcaa actccgcgct cactgtcaac agcaatatac cttctcgagc
     1921 cttctgttgg gttaacctga agaagtaatc ccagcaagtg tttccaagat gtgcaggcaa
     1981 cgattctgta aagtactgaa gcctcattca acatagtata tgtgctgccg aagcgagcac
     2041 ttaacaaggc ttgcggccgc tacttgtaca gctcgtccat gccgagagtg atcccggcgg
     2101 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
     2161 cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     2221 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
     2281 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     2341 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     2401 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     2461 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     2521 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt
     2581 aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     2641 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     2701 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     2761 cctcgccctt gctcaccatg gtggcgaccg gtagcgctag cataacttcg tataaagtat
     2821 cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc
     2881 aaagaataac ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc
     2941 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3001 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3061 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3121 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3181 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3241 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3301 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     3361 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3421 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     3481 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     3541 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     3601 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     3661 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     3721 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     3781 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     3841 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     3901 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     3961 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     4021 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     4081 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     4141 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4201 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4261 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     4321 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     4381 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4441 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4501 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4561 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4621 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     4681 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4741 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4801 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4861 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4921 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4981 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5041 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5101 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5161 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5221 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5281 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5341 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5401 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5461 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5521 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5581 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5641 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5701 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5761 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5821 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5881 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5941 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6001 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6061 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6121 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6181 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6241 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6301 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6361 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6421 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6481 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6541 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6601 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6661 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6721 ttgtgatgct cgtcaggggg gcggagccta tgga
//
