LOCUS       sequence-443553-        5282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  guideRNA targeting the mouse Rev-erb alpha (Nr1d1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5282)
  AUTHORS   Park I, Choi M, Kim J, Jang S, Kim D, Kim J, Choe Y, Geum D, Yu SW, 
            Choi JW, Moon C, Choe HK, Son GH, Kim K
  TITLE     Role of the circadian nuclear receptor REV-ERBalpha in dorsal raphe 
            serotonin synthesis in mood regulation.
  JOURNAL   Commun Biol. 2024 Aug 15;7(1):998. doi: 10.1038/s42003-024-06647-y.
  PUBMED    39147805
REFERENCE   2  (bases 1 to 5282)
  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: "Commun 
            Biol."; date: "2024-08-15"; volume: "7(1):998. doi"; pages: " 
            10.1038/s42003-024-06647-y"
COMMENT     Sequence Label: pAAV-U6-sgRev-erba-hSyn-mCherry-KASH
FEATURES             Location/Qualifiers
     source          1..5282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        417..492
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        526..973
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1005..1712
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIEEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHDEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1150..1168)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1418..1437
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2029..2505
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2228..2247)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2545..2685
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2545..2674
                     /label=AAV2 ITR
     rep_origin      2760..3215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2847..2866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3057..3078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3232..3251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3351..3373
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3411..3429)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3497..3601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3602..3670,3671..4462)
                     /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(3820..3839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4633..5221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5122..5141
                     /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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgttgcga ttgatgcgaa cgatgggttt
      421 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      481 ccgagtcggt gctttttttc tagactgcag agggccctgc gtatgagtgc aagtgggttt
      541 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      601 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
      661 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
      721 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
      781 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
      841 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
      901 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
      961 cctgagagcg cagtcgagaa ggtaccggat cctctagagt cgacatggtg agcaagggcg
     1021 aggaggataa catggccatc atcgaggagt tcatgcgctt caaggtgcac atggagggct
     1081 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1141 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1201 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1261 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1321 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1381 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1441 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1501 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1561 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1621 tcacctccca cgacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1681 actccaccgg cggcatggac gagctgtaca aggaattctc cggactcaga tctcgagagg
     1741 aggaggaggg gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga
     1801 gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc
     1861 tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg
     1921 ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca
     1981 cctaggatat caagcttatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     2041 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     2101 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     2161 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     2221 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     2281 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     2341 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     2401 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     2461 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     2521 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     2581 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     2641 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     2701 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     2761 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2821 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2881 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2941 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     3001 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3061 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     3121 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3181 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     3241 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3301 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3361 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     3421 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3541 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3601 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3661 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3721 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3781 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3841 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3901 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3961 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4021 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4081 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4141 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4201 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4261 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4321 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4381 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4441 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4501 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4741 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5281 gt
//
