LOCUS       sequence-190023-        5832 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of empty gRNA cassette and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5832)
  AUTHORS   Li Y, Kim J
  TITLE     Distinct roles of neuronal and microglial CB2 cannabinoid receptors 
            in the mouse hippocampus.
  JOURNAL   Neuroscience. 2017 Sep 6. pii: S0306-4522(17)30629-2. doi: 
            10.1016/j.neuroscience.2017.08.053.
  PUBMED    28888955
REFERENCE   2  (bases 1 to 5832)
  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: 
            "Neuroscience. 2017 Sep 6. pii: S0306-4522(17)30629-2. doi: 
            10.1016/j.neuroscience.2017.08.053."
COMMENT     Sequence Label: pAAV-U6-gRNA-CBh-mCherry
FEATURES             Location/Qualifiers
     source          1..5832
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        113..398
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(488..563)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(582..830)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(640..659)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(810..830)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   845..974
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1036..1624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1116..1135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1795..2586,2587..2655))
                     /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     2418..2437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2656..2760)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2828..2846
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2884..2906)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3006..3025
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3042..3497)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3179..3200)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3391..3410
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3572..3712
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3583..3712
                     /label=AAV2 ITR
     polyA_signal    3752..4228
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     4010..4029
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    4260..4848
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4366..4377
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4775..4795
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4873..5583)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5151..5170)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5420..5438
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5580..5589
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          5601..5828
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 cccccgcccc catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg
       61 gggagggggg ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa
      121 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
      181 gggcacaatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
      241 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
      301 gtcaatggaa agtccctatt ggcgttacta ttgacgtcaa tgggcggggg tcgttgggcg
      361 gtcagccagg cgggccattt accgtaagtt atgtaacggg tacctctaga atttgtctgc
      421 agaattggcg cacgcgctac ggactagcct tattttaact tgctatttct agctctaaaa
      481 caaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
      541 cttgctattt ctagctctaa aacagaagag ctcgctcttc cggtgtttcg tcctttccac
      601 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
      661 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
      721 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat ctctctaaca
      781 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc acgcgtgcgg
      841 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
      901 gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg
      961 cagctgcctg caggacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1021 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1081 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1141 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1201 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1261 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1321 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1381 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1441 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1501 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1561 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1621 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1681 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1741 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1801 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1861 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1921 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1981 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     2041 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     2101 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     2161 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     2221 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     2281 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     2341 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     2401 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     2461 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2521 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     2581 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2641 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2701 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2761 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     2821 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     2881 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2941 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     3001 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     3061 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga
     3121 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc
     3181 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     3241 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     3301 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3361 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa
     3421 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3481 gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga
     3541 tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc
     3601 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     3661 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
     3721 tcggtccgca cgtggttacc tacaaaatca gaaggacagg gaagggagca gtggttcacg
     3781 cctgtaatcc cagcaatttg ggaggccaag gtgggtagat cacctgagat taggagttgg
     3841 agaccagcct ggccaatatg gtgaaacccc gtctctacca aaaaaacaaa aattagctga
     3901 gcctggtcat gcatgcctgg aatcccaaca actcgggagg ctgaggcagg agaatcgctt
     3961 gaacccagga ggcggagatt gcagtgagcc aagattgtgc cactgcactc cagcttggtt
     4021 cccaatagac cccgcaggcc ctacaggttg tcttcccaac ttgccccttg ctccatacca
     4081 cccccctcca ccccataata ttatagaagg acacctagtc agacaaaatg atgcaactta
     4141 attttattag gacaaggctg gtgggcactg gagtggcaac ttccagggcc aggagaggca
     4201 ctggggaggg gtcacaggga tgccacccgt agatctctcg agcagcgctc ggtatcgatg
     4261 cggggaggcg gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa gacgcggaag
     4321 aggccgcaga gccggcagca ggccgcggga aggaaggtcc gctggattga gggccgaagg
     4381 gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca acataggcga gcagccaagg
     4441 aaaggacgat gatttccccg acaacaccac ggaattgtca gtgcccaaca gccgagcccc
     4501 tgtccagcag cgggcaaggc aggcggcgat gagttccgcc gtggcaatag ggagggggaa
     4561 agcgaaagtt ccggaaagga gctgacaggt ggtggcaatg ccccaaccag tgggggttgc
     4621 gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca acgggccaca actcctcata
     4681 aagagacagc aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat
     4741 agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt
     4801 aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattat cgataagctt
     4861 gatatcgaat tcttacttgt acagctcgtc catgccgccg gtggagtggc ggccctcggc
     4921 gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt ccaacttgat
     4981 gttgacgttg taggcgccgg gcagctgcac gggcttcttg gccttgtagg tggtcttgac
     5041 ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc tgcttgatct cgcccttcag
     5101 ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc cagcccatgg tcttcttctg
     5161 cattacgggg ccgtcggagg ggaagttggt gccgcgcagc ttcaccttgt agatgaactc
     5221 gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat
     5281 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc
     5341 ggggtgcttc acgtaggcct tggagccgta catgaactga ggggacagga tgtcccaggc
     5401 gaagggcagg gggccaccct tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg
     5461 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg
     5521 caccttgaag cgcatgaact ccttgatgat ggccatgtta tcctcctcgc ccttgctcac
     5581 catggtggca ccggtccaac ctgaaaaaaa gtgatttcag gcaggtgctc caggtaatta
     5641 aacattaata ccccaccaac caaccatccc ttaaaccctt acctcttgct cagctaatta
     5701 cagcccggag gagaagggcc gtcccgcccg ctcacctgtg ggagtaacgc ggtcagtcag
     5761 agccggggcg ggcggcgcga ggcggcggcg gagcggggca cggggcgaag gccgcgcgca
     5821 gcgactcccg cc
//
