LOCUS       sequence-193266-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA for mouse CB2 cannabinoid receptor and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  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 5927)
  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..5927
                     /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        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        508..793
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        795..1072
                     /label=chicken beta-actin promoter
     intron          1073..1301
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1313..1322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1319..2029
                     /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(1464..1482)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1732..1751
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2054..2642
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2107..2127)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2525..2536)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2674..3150
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2873..2892)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3190..3330
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3190..3319
                     /label=AAV2 ITR
     rep_origin      3405..3860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3492..3511)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3702..3723
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3877..3896)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3996..4018
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4056..4074)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4142..4246
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4247..4315,4316..5107)
                     /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(4465..4484)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5278..5866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5767..5786
                     /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 agaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtgacca acggctccaa cgggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
      541 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
      601 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      661 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
      721 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      781 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      841 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      901 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      961 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     1021 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     1081 tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1141 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1201 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     1261 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat
     1321 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1381 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1441 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1501 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1561 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1621 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1681 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     1741 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1801 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1861 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1921 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     1981 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag aattcgatat
     2041 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     2101 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     2161 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     2221 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     2281 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg gaactttcgc
     2341 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     2401 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     2461 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     2521 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     2581 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     2641 gcatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca
     2701 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     2761 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     2821 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     2881 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     2941 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     3001 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     3061 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     3121 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
     3181 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3241 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3301 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     3361 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3421 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3481 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3541 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3601 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3661 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3721 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     3781 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3841 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     3901 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3961 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4021 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4081 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4141 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4201 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4261 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4321 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4381 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4441 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4501 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4561 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4621 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4681 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4741 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4801 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4861 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4921 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4981 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5041 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5101 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5161 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5221 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5281 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5341 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5401 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5461 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5521 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5581 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5641 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5701 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5761 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5821 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5881 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
