LOCUS       sequence-148964-        6087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent knockdown of Scn9a (Nav1.7).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6087)
  AUTHORS   Branco T, Tozer A, Magnus CJ, Sugino K, Tanaka S, Lee AK, Wood JN, 
            Sternson SM
  TITLE     Near-Perfect Synaptic Integration by Nav1.7 in Hypothalamic Neurons 
            Regulates Body Weight.
  JOURNAL   Cell. 2016 Jun 16;165(7):1749-61. doi: 10.1016/j.cell.2016.05.019.
  PUBMED    27315482
REFERENCE   2  (bases 1 to 6087)
  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"; doi: 
            "10.1016/j.cell.2016.05"; journalName: "Cell"; date: "2016-06-16- 
            16"; volume: "165"; issue: "7"; pages: "1749-61"
COMMENT     Sequence Label: AAV-FLEX-EGFP-mir30(Scn9a)
FEATURES             Location/Qualifiers
     source          1..6087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2901..3178
                     /label=chicken beta-actin promoter
     primer_bind     3458..3480
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3542..3561
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(3597..3630)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(3597..3630)
                     /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(3649..3682)
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     ncRNA           complement(3753..3879)
                     /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(3907..3921)
                     /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(3949..4070)
                     /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(4134..4853)
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     regulatory      4850..4859
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4864..4897
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    4864..4897
                     /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    4942..4975
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    4989..5577
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5042..5062)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5460..5471)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5620..5741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5631..5650)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5685..5704
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5903..6032
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagatcgta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2701 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2761 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2821 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2881 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
     2941 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
     3001 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
     3061 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
     3121 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
     3181 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     3241 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     3301 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     3361 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
     3421 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     3481 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     3541 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tcctccataa
     3601 cttcgtatag gataccttat acgaagttat ctcagtttaa actaagccat aacttcgtat
     3661 agtacacatt atacgaagtt attgtttacc ggtacctgga ggaaaaaaaa aaggggagag
     3721 tcaggggtcg cgaattagtc ttccaattga aaaaagtgat ttaatttata ccattttaat
     3781 tcagctttgt aaaaatgtat caaagagata gcaaggtatt cagttttagt aaacaagata
     3841 attgctccta aagtagcccc ttgaattccg aggcagtagg catgggtggc atacgggtat
     3901 aaaatacatc tgtggcttca ctattttata cccgtatgcc acccgcgctc actgtcaaca
     3961 gcaatatacc ttctcgagcc ttctgttggg ttaacctgaa gaagtaatcc cagcaagtgt
     4021 ttccaagatg tgcaggcaac gattctgtaa agtactgaag cctcattcaa catagtatat
     4081 gtgctgccga agcgagcact taacaaggct tgatatccac tcacctgcaa ttgttacacc
     4141 cactcgtgca ggctgcccag gggcttgccc aggctggtca gctgggcgat ggcggtctcg
     4201 tgctgctcca cgaagccgcc gtcctccacg taggtcttct ccaggcggtg ctggatgaag
     4261 tggtactcgg ggaagtcctt caccacgccc ttgctcttca tcagggtgcg catgtggcag
     4321 ctgtagaact tgccgctgtt caggcggtac accaggatca cctggcccac cagcacgccg
     4381 tcgttcatgt acaccacctc gaagctgggc tgcaggccgg tgatggtctt cttcatcacg
     4441 gggccgtcgt tggggaagtt gcggcccttg tactccacgc ggtacacgaa catctcctcg
     4501 atcaggttga tgtcgctgcg gatctccacc aggccgccgt cctcgtagcg cagggtgcgc
     4561 tcgtacacga agccggcggg gaagctctgg atgaagaagt cgctgatgtc ctcggggtac
     4621 ttggtgaagg tgcggttgcc gtactggaag gcggggctca ggatgtcgaa ggcgaagggc
     4681 aggggggcgc ccttggtcac gcggatctgc accagctggt tgccgaacag gatgttgccc
     4741 ttgccgcagc cctccatggt gaacacgtgg ttgttcacca cgccctccag gttcaccttg
     4801 aagctcatga tctcctgcag gccggtgttc ttcaggatct gcttgctcac catggtggct
     4861 agcataactt cgtataaggt atcctatacg aagttatcca ttcaggctgt gtagcatcaa
     4921 tggcatggca caaacttagc cataacttcg tataatgtgt actatacgaa gttatcaagc
     4981 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     5041 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     5101 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     5161 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     5221 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     5281 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     5341 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     5401 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     5461 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     5521 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     5581 gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     5641 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     5701 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     5761 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     5821 acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat
     5881 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     5941 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     6001 cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg
     6061 ggagaggcgg tttgcgtatt gggcgct
//
