LOCUS       sequence-443551-        5283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  guideRNA targeting the mouse Pet-1 (Fev).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5283)
  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 5283)
  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-sgPet-1-hSyn-mCherry-KASH
FEATURES             Location/Qualifiers
     source          1..5283
                     /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        418..493
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        527..974
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1006..1713
                     /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(1151..1169)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1419..1438
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2030..2506
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2229..2248)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2546..2686
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2546..2675
                     /label=AAV2 ITR
     rep_origin      2761..3216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2848..2867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3058..3079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3233..3252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3352..3374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3412..3430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3498..3602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3603..3671,3672..4463)
                     /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(3821..3840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4634..5222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5123..5142
                     /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 accgccgcgc cacctcgtcc gggtcgggtt
      421 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      481 accgagtcgg tgcttttttt ctagactgca gagggccctg cgtatgagtg caagtgggtt
      541 ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc gacccactgg
      601 acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg gggaggggaa
      661 acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca gtgccttcgc
      721 ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg acgtcactcg
      781 ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc gccgccggcc
      841 cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac catctgcgct
      901 gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt
      961 gcctgagagc gcagtcgaga aggtaccgga tcctctagag tcgacatggt gagcaagggc
     1021 gaggaggata acatggccat catcgaggag ttcatgcgct tcaaggtgca catggagggc
     1081 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1141 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1201 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1261 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1321 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1381 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     1441 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1501 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1561 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1621 atcacctccc acgacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1681 cactccaccg gcggcatgga cgagctgtac aaggaattct ccggactcag atctcgagag
     1741 gaggaggagg ggacagacag caggatgccc cacctcgaca gccccggcag ctcccagccg
     1801 agacgctcct tcctctcaag ggtgatcagg gcagcgctac cgttgcagct gcttctgctg
     1861 ctgctgctgc tcctggcctg cctgctacct gcctctgaag atgactacag ctgcacccag
     1921 gccaacaact ttgcccgatc cttctacccc atgctgcggt acaccaacgg gccacctccc
     1981 acctaggata tcaagcttat cgataccgag cgctgctcga gagatctacg ggtggcatcc
     2041 ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag
     2101 ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
     2161 tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc
     2221 ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat
     2281 ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca
     2341 ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat
     2401 attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa
     2461 attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt
     2521 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     2581 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     2641 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     2701 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     2761 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2821 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2881 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2941 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3001 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3061 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     3121 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     3181 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3241 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3301 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3361 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3421 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3481 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3661 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3721 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3781 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3841 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3901 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3961 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4021 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4081 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4141 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4201 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4261 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4321 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4381 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4441 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4501 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5281 tgt
//
