LOCUS       sequence-325381-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the endocannabinoid sensor GRAB_eCB2.0 in Cre positive 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  AUTHORS   Dong A, He K, Dudok B, Farrell JS, Guan W, Liput DJ, Puhl HL, Cai R,
            Wang H, Duan J, Albarran E, Ding J, Lovinger DM, Li B, Soltesz I, Li
            Y
  TITLE     A fluorescent sensor for spatiotemporally resolved imaging of 
            endocannabinoid dynamics in vivo.
  JOURNAL   Nat Biotechnol. 2021 Nov 11. pii: 10.1038/s41587-021-01074-4. doi: 
            10.1038/s41587-021-01074-4.
  PUBMED    34764491
REFERENCE   2  (bases 1 to 6977)
  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: "Nat 
            Biotechnol. 2021 Nov 11. pii: 10.1038/s41587-021-01074-4. doi: 
            10.1038/s41587-021-01074-4."
COMMENT     Sequence Label: pAAV-hSyn-DIO-GRAB_eCB2.0
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(606..639)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(690..723)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1287..1306
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1546..1567
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1673..1694)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2863..2887)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2891..2950)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2947..2956
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2963..2996
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3047..3080
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3105..3693
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3158..3178)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3576..3587)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3725..4201
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3924..3943)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4241..4381
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4241..4370
                     /label=AAV2 ITR
     rep_origin      4456..4911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4543..4562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4753..4774
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4928..4947)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5047..5069
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5107..5125)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5193..5297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5298..5366,5367..6158)
                     /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(5516..5535)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6329..6917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6818..6837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtatgag gcggggtggg ggtgcctacc tgacgaccga
      181 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      241 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      301 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      361 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      421 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      481 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      541 aggagtcgtg tcgtgcctga gagcgcagtc gagaaaccgg tgccacctct agagtcgact
      601 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
      661 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
      721 tatggcgcat atgtcacaga gcctcggcag acgtgtctgt ggacacagac atggttacct
      781 tggcaatctt gaccgtgctc ttgatgcagc tttctgcggc cctgtgaaca ctggctgcat
      841 tgtttgcgtg tttgtgcagg cagtccgagt cccccatgct gttatccaga ggctgcgcag
      901 tgccttcaca agagggaaac atgctccgga aagcgtgtcg caggtcctta ctcctcagag
      961 catagatgat ggggttcacg gtggagttca gcaggcagag catggtgcag aatgcaaaca
     1021 ccgtcttaat gagcttgttc atcttcccaa agacatcata caccatgatt gcaagcagag
     1081 ggccccagca gatgatcaac accaccagga tcaggaccag ggtcttggct aacctaatgt
     1141 ccatgcgccg cccgcgccag cgtgctgctg ctccggtgtt gtactccagc ttgtgcccca
     1201 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1261 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1321 tgcgctcctg gatgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1381 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     1441 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccaacggtgg
     1501 catcgccctc accctcgccg cccacgctga acttgtggcc gtttacgtcg ccgtccagct
     1561 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc ctttctcacc atgctccctc
     1621 cggtaccgcc cttgtacagc tcgtccatgc cgaaagtgat cccggcggcg gtcacgaact
     1681 ccagcaggac catgtgatcg cgcttctcgt tggggtcttt cgaaagtttg gactgcacgc
     1741 tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt
     1801 agtggtaggc gagctgcacg ccgccgtcct cgatgttgtg gcggatgcag aagttcgcct
     1861 tgatgccgtt cttctgcttg tcggccttga tatagacgtt tcctccctcc tccaggtcca
     1921 gcgcgtcggt acgctgaatc atgcggacgg cgtggctgtg agccttccag agaatataca
     1981 tgtacgcata cacgatgaac agaagcagta cgctggtgac cccgatccag aacatcaggt
     2041 aggtttcatc aatgtgtggg aaaatgtctg agcaaacaga ttgcagtttc tcgcagttcc
     2101 agcccaggag aggcagcacg gcgatcacaa tggctatggt ccacatcagg caaaacgcca
     2161 ccacggcctt gggcctggtg acaatcctct tataggccag gggcctgtga atggatatgt
     2221 acctgtcgat ggctgtgagg aacaggctgc ccacggaggc agtgaaggag gccgtgaccc
     2281 cacccagttt gaacagaaac acgttgcggc tatctttgcg gtggaacacg tggaagtcaa
     2341 tgaagctgta gacaaaaatg acactcccca ggaggtctgc caccgccagg ctgccgatga
     2401 agtggtagga aggcctgcag cggaggctgc gggagtggag gatgacgcac agcaccagga
     2461 ggttctccag gaccgtgaag gtgcccagcg tgagggacag gactgcaatg gccagctgct
     2521 ggctggggtt caggaccatg aaacactcta tgtccatgaa gttctcccca cactggatgt
     2581 tctcctcatt ctccttgaag gacgagagag acttgttgta aaattctgta atgttcacct
     2641 ggtctgctgg gactagctgg gggttgtctc ccgcagtcat cttctcttgg aagggacttc
     2701 ccctaaagga agttaaaggg aatttctgtg ggaagtaccc taatttggat gccatgtcac
     2761 ctttgatgtc ttcgtactga atgtcatttg agcccacgta caggaggtca gtggtgatgg
     2821 tgcggaaggt ggtatctgca aggccatcta ggatcgactt ggtgccaact tttttgtaca
     2881 aacttgtgtc accagtggaa cctggaaccc agagcagcag tacccatagc aggagtgtgt
     2941 ctgtctccat ggtggcgcta gcataacttc gtataaagta tcctatacga agttatttgc
     3001 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     3061 tgtatgctat acgaagttat gaattcgata tcaagcttat cgataatcaa cctctggatt
     3121 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3181 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3241 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3301 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3361 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3421 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3481 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct
     3541 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3601 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3661 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat
     3721 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     3781 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     3841 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     3901 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     3961 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     4021 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     4081 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     4141 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     4201 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     4261 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4321 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4381 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4441 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4501 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4561 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4621 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4681 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4741 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4801 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4861 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4921 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4981 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5041 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5101 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5161 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5221 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5281 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5341 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5401 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5461 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5521 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5581 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5641 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5701 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5761 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5821 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5881 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5941 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6001 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6061 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6121 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6181 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6241 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6301 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6361 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6421 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6481 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6541 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6601 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6661 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6721 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6781 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6841 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6901 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6961 ggccttttgc tcacatg
//
