LOCUS       sequence-412537-        5995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of empty gRNA cassette and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5995)
  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 5995)
  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..5995
                     /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        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        577..862
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        864..1141
                     /label=chicken beta-actin promoter
     intron          1142..1369
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1381..1390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1387..2097
                     /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(1532..1550)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1800..1819
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2122..2710
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2175..2195)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2593..2604)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2742..3218
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2941..2960)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3258..3398
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3258..3387
                     /label=AAV2 ITR
     rep_origin      3473..3928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3560..3579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3770..3791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3945..3964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4064..4086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4124..4142)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4210..4314
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4315..4383,4384..5175)
                     /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(4533..4552)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5346..5934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5835..5854
                     /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 accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgtagcg
      541 cgtgcgccaa ttctgcagac aaattctaga ggtacccgtt acataactta cggtaaatgg
      601 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
      661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      781 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      841 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      901 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      961 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
     1021 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1081 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1141 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1201 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1261 tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt
     1321 attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt
     1381 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1441 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1501 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1561 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1621 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1681 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1741 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1801 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     1861 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1921 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1981 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     2041 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagaa
     2101 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     2161 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     2221 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     2281 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     2341 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccgga
     2401 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     2461 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     2521 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
     2581 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     2641 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     2701 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     2761 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     2821 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     2881 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     2941 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     3001 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     3061 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     3121 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     3181 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     3241 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3301 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3361 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3421 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3481 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3541 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3601 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3661 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3721 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3781 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     3841 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3901 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     3961 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4021 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4081 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4141 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4201 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4261 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4321 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4381 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4441 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4501 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4561 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4621 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4681 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4741 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4801 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4861 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4921 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4981 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5041 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5101 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5161 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5221 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5281 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5341 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5401 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5461 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5521 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5581 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5641 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5701 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5761 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5821 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5881 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5941 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
