LOCUS       sequence-296620-        6581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the adenosine sensor GRAB_Ado1.0 in Cre positive cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6581)
  AUTHORS   Peng W, Wu Z, Song K, Zhang S, Li Y, Xu M
  TITLE     Regulation of sleep homeostasis mediator adenosine by basal 
            forebrain glutamatergic neurons.
  JOURNAL   Science. 2020 Sep 4;369(6508). pii: 369/6508/eabb0556. doi: 
            10.1126/science.abb0556.
  PUBMED    32883833
REFERENCE   2  (bases 1 to 6581)
  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: "Science."; 
            date: "2020-09-4"; volume: "369(6508). pii: 369/6508/eabb0556. doi";
            pages: " 10.1126/science.abb0556"
COMMENT     Sequence Label: pAAV-EFS-DIO-GRAB_Ado1.0
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..142
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   13..142
                     /label=AAV2 ITR
     promoter        163..374
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(393..426)
                     /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(477..510)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1227..1246
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1486..1507
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1613..1634)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2692..2716)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2720..2779)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2776..2785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2792..2825
                     /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    2876..2909
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2934..3522
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2987..3007)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3405..3416)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3554..4030
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3753..3772)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4070..4210
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4070..4199
                     /label=AAV2 ITR
     rep_origin      4285..4740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4372..4391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4582..4603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4757..4776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4876..4898
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4936..4954)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5022..5126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5127..5195,5196..5987)
                     /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(5345..5364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
       61 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      121 aactccatca ctaggggttc ctgcggccgc acgcgtaagc ttgggcagag cgcacatcgc
      181 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
      241 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
      301 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
      361 ccgccagaac acagtctaga gtcgactccg gaataacttc gtataggata ctttatacga
      421 agttatgcag aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttaataa
      481 cttcgtatag catacattat acgaagttat ggcgcatatg tcaggacact cctgctccat
      541 cctgggccag ggggtcatct aggccagggg gctctgggca cactcccttg agctcatggc
      601 taaggagctc cacgtctggg aggcccgtgt tcccctggga ctcttgggca ctccctccac
      661 tcaccagccc cagggcgtag ccattgggcc tccgctcagg gtggggagca ctgccgttgg
      721 cccacactcc tggcgggtgg ccgttgagac ggaggctgac ctgctctccg tcactgccat
      781 gagctgccaa gacccgggca ctggtgccag ctgccttgaa aggttcttgc tgcctcagga
      841 cgtggctgcg aatgatcttg cggaaggtct ggcggaactc gcggatacgg taggcgtaga
      901 tgaagggatt cacaaccgaa ttggtgtggg agaggacgat ggccaggtac atgagccaga
      961 gaggggcgtg gctgcagtcg gggcagaaga aagtgaagca gttgatgatg tgtaggggca
     1021 gccagcagag ggcaaagagc cccacaatga tggccagtga cttggcagca tggagctcgc
     1081 ggttctgccg cccgcgccag cgtgctgctg ctccggtgtt gtactccagc ttgtgcccca
     1141 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1201 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1261 tgcgctcctg gatgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1321 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     1381 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     1441 catcgccctc accctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     1501 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc ctttctcacc atgctccctc
     1561 cggtaccgcc cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact
     1621 ccagcaggac catgtgatcg cgcttctcgt tggggtcttt cgaaagtttg gactgcacgc
     1681 tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt
     1741 agtggtaggc gagctgcacg ccgccgtcct cgatgttgtg gcggatgtgg aagttcgcct
     1801 tgatgccgtt cttctgcttg tcggccttga tatagacgtt tcctccctcc tccaggtcca
     1861 gcgcgtcggt gtcgcgcggc ccggccggcg cgggctcgcc aggggcgccg ttgagctggg
     1921 tgggcagcgg ttcggcctct gcgccccccg ggcccgcgct gcgctcgggg cccagaccgt
     1981 tgggcctgcg ctcggtgccc cccggcggcg cggcgacggc gtccggaccc cggcggctgg
     2041 gaggctggct ctccatctgc ttcagctgtc gtcgcgccgc caggaagatc cgcaaataga
     2101 cacccagcat gagcagcagg ggcaccagca cacaggcaaa gaagttgaag tacaccatgt
     2161 agttcatggg gaccacatcc tcaaagagac aggccacttg gccctccccg cagccctggg
     2221 agtggttctt gccctccttt ggctgaccgc agttgttcca acctagcatg ggagtcaggc
     2281 cgatggcaaa cgacagcacc cagcagatgg caatgatgcc cttagccctc gtgccggtca
     2341 ccaagccatt gtaccggagc gggatgcgga tggcaatgta gcggtcaatg gcgatggcca
     2401 ggagactgaa gatggagctc tgcgtgagga ccaggacgaa gcaggcaatg aagaggcagc
     2461 cgtggcaggc agcgcagaac ccggtgctga tggtgatggc aaaggggatg gcgagcacac
     2521 ccactgcgat gtcggccgcc gccagtgaca ccacaaagta gttggtgacg ttctgcaggt
     2581 tgctgttgag ccacacggcc cagcacacca gcacattgcc caggatggcc agcacagcaa
     2641 tggccagctc caccgtgatg tacaccgagg agcccatgat gggaccggtg gtgccaactt
     2701 ttttgtacaa acttgtgtca ccagtggaac ctggaaccca gagcagcagt acccatagca
     2761 ggagtgtgtc tgtctccatg gtggcgctag cataacttcg tataaagtat cctatacgaa
     2821 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     2881 ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac
     2941 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3001 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3061 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3121 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3181 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3241 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3301 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg
     3361 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3421 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     3481 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc
     3541 tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga
     3601 agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc
     3661 tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca
     3721 agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt
     3781 ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca
     3841 gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt
     3901 ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt
     3961 gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt
     4021 ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta
     4081 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4141 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4201 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4261 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4321 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4381 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4441 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4501 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4561 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4621 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4681 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     4741 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     4801 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4861 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4921 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     4981 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5041 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5101 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5161 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5221 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5281 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5341 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5401 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5461 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5521 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5581 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5641 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5701 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5761 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5821 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5881 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5941 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6001 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6061 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6121 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6181 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6241 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6301 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6361 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6421 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6481 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6541 agcgtgagct atgagaaagc gccacgcttc ccgaagggag a
//
