LOCUS       sequence-325155-        6700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the endocannabinoid sensor GRAB_eCB2.0 in FLP positive 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6700)
  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 6700)
  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-EFS-fDIO-GRAB_eCB2.0
FEATURES             Location/Qualifiers
     source          1..6700
                     /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..356
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    373..420
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    443..476
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     1044..1063
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1303..1324
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1430..1451)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2620..2644)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2648..2707)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2704..2713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2720..2767)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(2769..2802)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    2840..3428
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2893..2913)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3311..3322)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3447..3923
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3646..3665)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3963..4103
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3963..4092
                     /label=AAV2 ITR
     rep_origin      4178..4633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4265..4284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4475..4496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4650..4669)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4769..4791
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4829..4847)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4915..5019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5020..5088,5089..5880)
                     /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(5238..5257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6051..6639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6540..6559
                     /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 gcgtgggcag agcgcacatc gcccacagtc cccgagaagt
      181 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      241 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      301 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagtgga
      361 gaattcggat ccgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
      421 ttaattaaga agttcctatt ccgaagttcc tattcttcaa atagtatagg aacttcggcg
      481 cgccccatgg tcacagagcc tcggcagacg tgtctgtgga cacagacatg gttaccttgg
      541 caatcttgac cgtgctcttg atgcagcttt ctgcggccct gtgaacactg gctgcattgt
      601 ttgcgtgttt gtgcaggcag tccgagtccc ccatgctgtt atccagaggc tgcgcagtgc
      661 cttcacaaga gggaaacatg ctccggaaag cgtgtcgcag gtccttactc ctcagagcat
      721 agatgatggg gttcacggtg gagttcagca ggcagagcat ggtgcagaat gcaaacaccg
      781 tcttaatgag cttgttcatc ttcccaaaga catcatacac catgattgca agcagagggc
      841 cccagcagat gatcaacacc accaggatca ggaccagggt cttggctaac ctaatgtcca
      901 tgcgccgccc gcgccagcgt gctgctgctc cggtgttgta ctccagcttg tgccccagga
      961 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
     1021 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
     1081 gctcctggat gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     1141 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg
     1201 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgcca acggtggcat
     1261 cgccctcacc ctcgccgccc acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     1321 ccaggatggg caccaccccg gtgaacagct cctcgccctt tctcaccatg ctccctccgg
     1381 taccgccctt gtacagctcg tccatgccga aagtgatccc ggcggcggtc acgaactcca
     1441 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga aagtttggac tgcacgctca
     1501 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1561 ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg gatgcagaag ttcgccttga
     1621 tgccgttctt ctgcttgtcg gccttgatat agacgtttcc tccctcctcc aggtccagcg
     1681 cgtcggtacg ctgaatcatg cggacggcgt ggctgtgagc cttccagaga atatacatgt
     1741 acgcatacac gatgaacaga agcagtacgc tggtgacccc gatccagaac atcaggtagg
     1801 tttcatcaat gtgtgggaaa atgtctgagc aaacagattg cagtttctcg cagttccagc
     1861 ccaggagagg cagcacggcg atcacaatgg ctatggtcca catcaggcaa aacgccacca
     1921 cggccttggg cctggtgaca atcctcttat aggccagggg cctgtgaatg gatatgtacc
     1981 tgtcgatggc tgtgaggaac aggctgccca cggaggcagt gaaggaggcc gtgaccccac
     2041 ccagtttgaa cagaaacacg ttgcggctat ctttgcggtg gaacacgtgg aagtcaatga
     2101 agctgtagac aaaaatgaca ctccccagga ggtctgccac cgccaggctg ccgatgaagt
     2161 ggtaggaagg cctgcagcgg aggctgcggg agtggaggat gacgcacagc accaggaggt
     2221 tctccaggac cgtgaaggtg cccagcgtga gggacaggac tgcaatggcc agctgctggc
     2281 tggggttcag gaccatgaaa cactctatgt ccatgaagtt ctccccacac tggatgttct
     2341 cctcattctc cttgaaggac gagagagact tgttgtaaaa ttctgtaatg ttcacctggt
     2401 ctgctgggac tagctggggg ttgtctcccg cagtcatctt ctcttggaag ggacttcccc
     2461 taaaggaagt taaagggaat ttctgtggga agtaccctaa tttggatgcc atgtcacctt
     2521 tgatgtcttc gtactgaatg tcatttgagc ccacgtacag gaggtcagtg gtgatggtgc
     2581 ggaaggtggt atctgcaagg ccatctagga tcgacttggt gccaactttt ttgtacaaac
     2641 ttgtgtcacc agtggaacct ggaacccaga gcagcagtac ccatagcagg agtgtgtctg
     2701 tctccatggt ggcgctagcg aagttcctat actttctaga gaataggaac ttcggaatag
     2761 gaacttcgga agttcctata ctatttgaag aataggaact tcggaatagg aacttcaatt
     2821 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2881 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2941 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3001 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3061 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3121 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3181 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3241 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3301 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3361 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3421 ctccccgcat cgataccgag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     3481 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     3541 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     3601 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     3661 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     3721 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     3781 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     3841 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     3901 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     3961 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4021 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4081 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4141 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4201 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4261 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4321 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4381 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4441 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4501 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4561 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4621 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4681 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4741 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4801 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4861 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4921 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4981 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5041 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5101 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5161 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5221 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5281 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5341 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5401 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5461 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5521 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5581 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5641 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5701 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5761 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5821 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5881 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5941 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6001 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6061 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6181 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6661 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
