LOCUS       sequence-190025-        5696 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of gRNA for mouse CB2 cannabinoid receptor and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5696)
  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 5696)
  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-CB2gRNA-CBh-mCherry
FEATURES             Location/Qualifiers
     source          1..5696
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        65..350
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(369..444)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(465..713)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(523..542)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(693..713)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   728..857
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(919..1507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(999..1018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1678..2469,2470..2538))
                     /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     2301..2320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2539..2643)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2711..2729
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2767..2789)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2889..2908
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2925..3380)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3062..3083)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3274..3293
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3455..3595
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3466..3595
                     /label=AAV2 ITR
     polyA_signal    3635..4111
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     3893..3912
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    4143..4731
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4249..4260
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4658..4678
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4756..5466)
                     /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(5034..5053)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5303..5321
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5463..5472
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg gggctcacct
       61 cgaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
      121 aggtcatgta ctgggcacaa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
      181 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
      241 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tattgacgtc aatgggcggg
      301 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg ggtacctcta
      361 gaaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta
      421 acttgctatt tctagctcta aaacccgttg gagccgttgg tcacggtgtt tcgtcctttc
      481 cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag
      541 tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt
      601 cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat tatctctcta
      661 acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcacgcgtg
      721 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
      781 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggcg gcctcagtga gcgagcgagc
      841 gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      901 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      961 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1021 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1081 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1141 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1201 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1261 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1321 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1381 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1441 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1501 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1561 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1621 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1681 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1741 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1801 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1861 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1921 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1981 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2041 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2101 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2161 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2221 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2281 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2341 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2401 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2461 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2521 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2581 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2641 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     2701 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     2761 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     2821 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     2881 taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa
     2941 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac
     3001 cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt
     3061 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     3121 aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg
     3181 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     3241 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3301 taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3361 tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac
     3421 agatgcgtaa ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg cgctcgctcg
     3481 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca
     3541 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc
     3601 cgctcggtcc gcacgtggtt acctacaaaa tcagaaggac agggaaggga gcagtggttc
     3661 acgcctgtaa tcccagcaat ttgggaggcc aaggtgggta gatcacctga gattaggagt
     3721 tggagaccag cctggccaat atggtgaaac cccgtctcta ccaaaaaaac aaaaattagc
     3781 tgagcctggt catgcatgcc tggaatccca acaactcggg aggctgaggc aggagaatcg
     3841 cttgaaccca ggaggcggag attgcagtga gccaagattg tgccactgca ctccagcttg
     3901 gttcccaata gaccccgcag gccctacagg ttgtcttccc aacttgcccc ttgctccata
     3961 ccacccccct ccaccccata atattataga aggacaccta gtcagacaaa atgatgcaac
     4021 ttaattttat taggacaagg ctggtgggca ctggagtggc aacttccagg gccaggagag
     4081 gcactgggga ggggtcacag ggatgccacc cgtagatctc tcgagcagcg ctcggtatcg
     4141 atgcggggag gcggcccaaa gggagatccg actcgtctga gggcgaaggc gaagacgcgg
     4201 aagaggccgc agagccggca gcaggccgcg ggaaggaagg tccgctggat tgagggccga
     4261 agggacgtag cagaaggacg tcccgcgcag aatccaggtg gcaacatagg cgagcagcca
     4321 aggaaaggac gatgatttcc ccgacaacac cacggaattg tcagtgccca acagccgagc
     4381 ccctgtccag cagcgggcaa ggcaggcggc gatgagttcc gccgtggcaa tagggagggg
     4441 gaaagcgaaa gttccggaaa ggagctgaca ggtggtggca atgccccaac cagtgggggt
     4501 tgcgtcagca aacacagtgc acaccacgcc acgttgcctg acaacgggcc acaactcctc
     4561 ataaagagac agcaaccagg atttatacaa ggaggagaaa atgaaagcca tacgggaagc
     4621 aatagcatga tacaaaggca ttaaagcagc gtatccacat agcgtaaaag gagcaacata
     4681 gttaagaata ccagtcaatc tttcacaaat tttgtaatcc agaggttgat tatcgataag
     4741 cttgatatcg aattcttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
     4801 ggcgcgttcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt
     4861 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt
     4921 gacctcagcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt
     4981 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt
     5041 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa
     5101 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt
     5161 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc
     5221 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca
     5281 ggcgaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     5341 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat
     5401 gtgcaccttg aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct
     5461 caccatggtg gcaccggtcc aacctgaaaa aaagtgattt caggcaggtg ctccaggtaa
     5521 ttaaacatta ataccccacc aaccaaccat cccttaaacc cttacctctt gctcagctaa
     5581 ttacagcccg gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt
     5641 cagagccggg gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcc
//
