LOCUS       sequence-305180-        6795 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 6795)
  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 6795)
  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..6795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      675..1263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1164..1183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1325..1454
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1475..1686
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(1705..1738)
                     /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(1789..1822)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2539..2558
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2798..2819
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2925..2946)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4004..4028)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(4032..4091)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      4088..4097
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4104..4137
                     /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    4188..4221
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4246..4834
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4299..4319)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4717..4728)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4866..5342
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5065..5084)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5382..5522
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5382..5511
                     /label=AAV2 ITR
     rep_origin      5597..6052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5684..5703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5894..5915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6069..6088)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6188..6210
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6248..6266)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6334..6438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6439..6507,6508..6795,1..504)
                     /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(6657..6676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
       61 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      121 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      181 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      241 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      301 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      361 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      421 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      481 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
      541 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
      601 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      661 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      721 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      781 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      841 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      901 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      961 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1021 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1081 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1141 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1201 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1261 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1321 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     1381 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     1441 cactaggggt tcctgcggcc gcacgcgtaa gcttgggcag agcgcacatc gcccacagtc
     1501 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     1561 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     1621 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     1681 acacagtcta gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc
     1741 agaatggtag ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat
     1801 agcatacatt atacgaagtt atggcgcata tgtcaggaca ctcctgctcc atcctgggcc
     1861 agggggtcat ctaggccagg gggctctggg cacactccct tgagctcatg gctaaggagc
     1921 tccacgtctg ggaggcccgt gttcccctgg gactcttggg cactccctcc actcaccagc
     1981 cccagggcgt agccattggg cctccgctca gggtggggag cactgccgtt ggcccacact
     2041 cctggcgggt ggccgttgag acggaggctg acctgctctc cgtcactgcc atgagctgcc
     2101 aagacccggg cactggtgcc agctgccttg aaaggttctt gctgcctcag gacgtggctg
     2161 cgaatgatct tgcggaaggt ctggcggaac tcgcggatac ggtaggcgta gatgaaggga
     2221 ttcacaaccg aattggtgtg ggagaggacg atggccaggt acatgagcca gagaggggcg
     2281 tggctgcagt cggggcagaa gaaagtgaag cagttgatga tgtgtagggg cagccagcag
     2341 agggcaaaga gccccacaat gatggccagt gacttggcag catggagctc gcggttctgc
     2401 cgcccgcgcc agcgtgctgc tgctccggtg ttgtactcca gcttgtgccc caggatgttg
     2461 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg
     2521 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     2581 tggatgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     2641 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     2701 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     2761 tcaccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     2821 atgggcacca ccccggtgaa cagctcctcg ccctttctca ccatgctccc tccggtaccg
     2881 cccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     2941 accatgtgat cgcgcttctc gttggggtct ttcgaaagtt tggactgcac gctcaggtag
     3001 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtag
     3061 gcgagctgca cgccgccgtc ctcgatgttg tggcggatgt ggaagttcgc cttgatgccg
     3121 ttcttctgct tgtcggcctt gatatagacg tttcctccct cctccaggtc cagcgcgtcg
     3181 gtgtcgcgcg gcccggccgg cgcgggctcg ccaggggcgc cgttgagctg ggtgggcagc
     3241 ggttcggcct ctgcgccccc cgggcccgcg ctgcgctcgg ggcccagacc gttgggcctg
     3301 cgctcggtgc cccccggcgg cgcggcgacg gcgtccggac cccggcggct gggaggctgg
     3361 ctctccatct gcttcagctg tcgtcgcgcc gccaggaaga tccgcaaata gacacccagc
     3421 atgagcagca ggggcaccag cacacaggca aagaagttga agtacaccat gtagttcatg
     3481 gggaccacat cctcaaagag acaggccact tggccctccc cgcagccctg ggagtggttc
     3541 ttgccctcct ttggctgacc gcagttgttc caacctagca tgggagtcag gccgatggca
     3601 aacgacagca cccagcagat ggcaatgatg cccttagccc tcgtgccggt caccaagcca
     3661 ttgtaccgga gcgggatgcg gatggcaatg tagcggtcaa tggcgatggc caggagactg
     3721 aagatggagc tctgcgtgag gaccaggacg aagcaggcaa tgaagaggca gccgtggcag
     3781 gcagcgcaga acccggtgct gatggtgatg gcaaagggga tggcgagcac acccactgcg
     3841 atgtcggccg ccgccagtga caccacaaag tagttggtga cgttctgcag gttgctgttg
     3901 agccacacgg cccagcacac cagcacattg cccaggatgg ccagcacagc aatggccagc
     3961 tccaccgtga tgtacaccga ggagcccatg atgggaccgg tggtgccaac ttttttgtac
     4021 aaacttgtgt caccagtgga acctggaacc cagagcagca gtacccatag caggagtgtg
     4081 tctgtctcca tggtggcgct agcataactt cgtataaagt atcctatacg aagttatttg
     4141 ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata
     4201 atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat
     4261 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     4321 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     4381 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     4441 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     4501 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     4561 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     4621 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     4681 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     4741 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     4801 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     4861 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     4921 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     4981 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     5041 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     5101 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     5161 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     5221 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     5281 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     5341 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     5401 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     5461 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     5521 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     5581 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5641 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     5701 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5761 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5821 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5881 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     5941 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     6001 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     6061 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     6121 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6181 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     6241 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6301 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6361 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6421 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6481 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6541 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6601 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6661 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6721 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6781 atgacagtaa gagaa
//
