LOCUS       sequence-325383-        6977 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express the endocannabinoid non-responsive 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 Apr 14, 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_eCB2.0mut
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   43..172
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(647..680)
                     /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(731..764)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1328..1347
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1587..1608
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1714..1735)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2904..2928)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2929..2991)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     regulatory      2988..2997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3004..3037
                     /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    3088..3121
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3146..3734
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3199..3219)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3617..3628)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3766..4242
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3965..3984)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4282..4422
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4282..4411
                     /label=AAV2 ITR
     rep_origin      4497..4952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4584..4603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4794..4815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4969..4988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5088..5110
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5148..5166)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5234..5338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5339..5407,5408..6199)
                     /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(5557..5576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6370..6958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6859..6878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
       61 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
      121 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
      181 acgcgtatga ggcggggtgg gggtgcctac ctgacgaccg accccgaccc actggacaag
      241 cacccaaccc ccattcccca aattgcgcat cccctatcag agagggggag gggaaacagg
      301 atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc ttcgcccccg
      361 cctggcggcg cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc actcgccggt
      421 cccccgcaaa ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc
      481 ggaccgcacc acgcgaggcg cgagataggg gggcacgggc gcgaccatct gcgctgcggc
      541 gccggcgact cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg
      601 agagcgcagt cgagaaaccg gtgccacctc tagagtcgac tccggaataa cttcgtatag
      661 gatactttat acgaagttat gcagaatggt agctggattg tagctgctat tagcaatatg
      721 aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgca tatgtcacag
      781 agcctcggca gacgtgtctg tggacacaga catggttacc ttggcaatct tgaccgtgct
      841 cttgatgcag ctttctgcgg ccctgtgaac actggctgca ttgtttgcgt gtttgtgcag
      901 gcagtccgag tcccccatgc tgttatccag aggctgcgca gtgccttcac aagagggaaa
      961 catgctccgg aaagcgtgtc gcaggtcctt actcctcaga gcatagatga tggggttcac
     1021 ggtggagttc agcaggcaga gcatggtgca gaatgcaaac accgtcttaa tgagcttgtt
     1081 catcttccca aagacatcat acaccatgat tgcaagcaga gggccccagc agatgatcaa
     1141 caccaccagg atcaggacca gggtcttggc taacctaatg tccatgcgcc gcccgcgcca
     1201 gcgtgctgct gctccggtgt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     1261 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     1321 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggatgtagcc
     1381 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa
     1441 gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     1501 ggtggtgcag atgaacttca gggtcagctt gccaacggtg gcatcgccct caccctcgcc
     1561 gcccacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     1621 cccggtgaac agctcctcgc cctttctcac catgctccct ccggtaccgc ccttgtacag
     1681 ctcgtccatg ccgaaagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc
     1741 gcgcttctcg ttggggtctt tcgaaagttt ggactgcacg ctcaggtagt ggttgtcggg
     1801 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtagg cgagctgcac
     1861 gccgccgtcc tcgatgttgt ggcggatgca gaagttcgcc ttgatgccgt tcttctgctt
     1921 gtcggccttg atatagacgt ttcctccctc ctccaggtcc agcgcgtcgg tacgctgaat
     1981 catgcggacg gcgtggctgt gagccttcca gagaatatac atgtacgcat acacgatgaa
     2041 cagaagcagt acgctggtga ccccgatcca gaacatcagg taggtttcat caatgtgtgg
     2101 gaaaatgtct gagcaaacag attgcagttt ctcgcagttc cagcccagga gaggcagcac
     2161 ggcgatcaca atggctatgg tccacatcag gcaaaacgcc accacggcct tgggcctggt
     2221 gacaatcctc ttataggcca ggggcctgtg aatggatatg tacctgtcga tggctgtgag
     2281 gaacaggctg cccacggagg cagtgaagga ggccgtgacc ccacccagtt tgaacagaaa
     2341 cacgttgcgg ctatctttgc ggtggaacac gtgggcgtca atgaagctgt agacaaaaat
     2401 gacactcccc aggaggtctg ccaccgccag gctgccgatg aagtggtagg aaggcctgca
     2461 gcggaggctg cgggagtgga ggatgacgca cagcaccagg aggttctcca ggaccgtgaa
     2521 ggtgcccagc gtgagggaca ggactgcaat ggccagctgc tggctggggt tcaggaccat
     2581 gaaacactct atgtccatga agttctcccc acactggatg ttctcctcat tctccttgaa
     2641 ggacgagaga gacttgttgt aaaattctgt aatgttcacc tggtctgctg ggactagctg
     2701 ggggttgtct cccgcagtca tcttctcttg gaagggactt cccctaaagg aagttaaagg
     2761 gaatttctgt gggaagtacc ctaatttgga tgccatgtca cctttgatgt cttcgtactg
     2821 aatgtcattt gagcccacgt acaggaggtc agtggtgatg gtgcggaagg tggtatctgc
     2881 aaggccatct aggatcgact tggtgccaac ttttttgtac aaacttgtgt caccagtgga
     2941 acctggaacc cagagcagca gtacccatag caggagtgtg tctgtctcca tggtggcgct
     3001 agcataactt cgtataaagt atcctatacg aagttatttg ccttaaccca gaaattatca
     3061 ctgttattct ttagaatggt gcaaagaata acttcgtata atgtatgcta tacgaagtta
     3121 tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3181 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3241 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3301 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3361 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3421 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3481 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3541 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     3601 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3661 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3721 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     3781 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     3841 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     3901 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     3961 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     4021 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     4081 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     4141 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     4201 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     4261 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4321 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4381 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4441 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4501 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4561 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4621 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4681 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4741 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4801 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4861 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4921 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4981 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5041 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5101 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5161 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5221 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5281 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5341 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5401 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5461 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5521 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5581 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5641 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5701 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5761 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5821 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5881 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5941 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6001 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6061 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6121 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6181 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6241 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6301 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6361 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6421 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6481 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6541 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6601 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6661 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6721 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6781 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6841 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6901 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6961 cgccagcaac gcggcct
//
