pAAV-GfaABC1D-Asp-RGEPO1

LOCUS       pAAV-GfaABC1D-Asp-RGEPO1               6571 bp DNA     circular SYN 26-MAR-2024
DEFINITION  Express the adenosine sensor GRAB_Ado1.0 in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6571)
  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 6571)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2026 from SnapGene 6.0.2
            https://www.biofeng.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"
FEATURES             Location/Qualifiers
     source          1..6571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        855..1356
                     /label=GfaABC1D
     misc_feature    931..936
                     /label=SDS
     misc_feature    998..1003
                     /label=SDS
     primer_bind     complement(1200..1211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    1306..1311
                     /label=SDS
     primer_bind     1505..1526
                     /label=GfaABC1D-F
     intron          1645..1777
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1760..1779
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1822..1840
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1837..1874
                     /label=Gfa-NmA-F2
     primer_bind     complement(1839..1858)
                     /label=R1
     regulatory      1857..1862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1863..1934
                     /codon_start=1
                     /label=Asp-tag
                     /translation="MASSLYSFLLALSIVYIFVAPTHS"
     CDS             1935..1961
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     1957..2001
                     /label=PD-F1
     CDS             1983..2435
                     /codon_start=1
                     /label=N-mApple
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTARLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMSF
                     EDGGIIHVNQDSSLQDGVFIYKVKLLGTNFPPDGPVMQKKTMGWEAS"
     primer_bind     1983..2000
                     /label=N-mapple-F2
     primer_bind     1983..1995
                     /label=Intra-F1
     primer_bind     1983..1995
                     /label=Intra-cag-F1
     primer_bind     2409..2456
                     /label=FR-1-3-F
     primer_bind     2409..2430
                     /label=FR-Y-Ra-F
     primer_bind     complement(2418..2438)
                     /label=mApple-N-R1
     primer_bind     complement(2422..2435)
                     /label=N-mapple-R
     primer_bind     complement(2424..2442)
                     /label=FR-1-3-R
     primer_bind     2432..2471
                     /label=PA-S9A-F1
     primer_bind     2433..2465
                     /label=PA-F6A-F1
     CDS             2436..2438
                     /codon_start=1
                     /label=LINKER1
                     /translation="P"
     primer_bind     complement(2437..2486)
                     /label=F6A-R1
     CDS             2439..2870
                     /codon_start=1
                     /label=YgaU_Hydrothermal
                     /translation="GIWDFVKSAGRKIGIGSEEAAPAADDLKKELDSYKLGTDDVKVEV
                     VGDKAVITGNVADQSILEKAIVAVGNTLGISKVESNVEVPDAKEPVFHTVKKGDTLWAI
                     ASKQYGNVSKYNAIFEANKPMLSHPDKIYPGQVLRIPDLG"
     primer_bind     complement(2442..2486)
                     /label=V7A-R1
     primer_bind     complement(2445..2486)
                     /label=K8A-R1
     primer_bind     complement(2449..2486)
                     /label=S9A-R1
     misc_feature    2451..2471
                     /label=Mutant
     primer_bind     complement(2456..2486)
                     /label=G11A-R1
     primer_bind     complement(2456..2486)
                     /label=R12A-R1
     primer_bind     2472..2489
                     /label=0811-F2
     primer_bind     2629..2661
                     /label=PA-T73A-F1
     primer_bind     complement(2633..2684)
                     /label=G71A-R1
     primer_bind     2633..2667
                     /label=PA-G75A-F1
     primer_bind     complement(2633..2650)
                     /label=0905-R2
     primer_bind     complement(2634..2684)
                     /label=N72A-R1
     primer_bind     complement(2637..2684)
                     /label=T73A-R1
     misc_feature    2646..2669
                     /label=Mmutant
     primer_bind     complement(2648..2684)
                     /label=G75A-R1
     primer_bind     complement(2649..2684)
                     /label=I76A-R1
     primer_bind     complement(2651..2684)
                     /label=S77A-R1
     primer_bind     complement(2656..2685)
                     /label=K78A-R1
     primer_bind     complement(2658..2684)
                     /label=L74A-R1
     primer_bind     2670..2688
                     /label=0811-F1
     primer_bind     complement(2719..2737)
                     /label=YgaU-R1
     primer_bind     complement(2762..2786)
                     /label=G113V-R
     primer_bind     complement(2847..2863)
                     /label=Ec-kbp-R
     primer_bind     2871..2889
                     /label=mApple-C-F1
     CDS             2871..2879
                     /codon_start=1
                     /label=LINKER
                     /translation="SAC"
     CDS             2880..3137
                     /codon_start=1
                     /label=C-mApple
                     /translation="VSEWMYPEDGALKSEIRKGLRLKDGGHYAAEVKTTYKAKKPVQLP
                     GAYIVDIELDILSHNEDYTIVEQCERAVGRHPTGGTDALYK"
     primer_bind     2880..2900
                     /label=C-mApple-F2
     primer_bind     complement(3070..3089)
                     /label=FR-R
     primer_bind     complement(3119..3158)
                     /label=PD-R1
     primer_bind     3123..3139
                     /label=0821_KA_FRCaMPi_F
     primer_bind     3138..3158
                     /label=MYC-F1
     CDS             3147..3176
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3180..3326
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(3307..3349)
                     /label=CmA-WPRE-R2
     primer_bind     3330..3349
                     /label=WPRE-F1
     misc_feature    3348..3936
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3401..3421)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     misc_feature    3484..3489
                     /label=SDS
     CDS             complement(3819..3830)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3968..4444
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4167..4186)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    4224..4229
                     /label=SDS
     repeat_region   4484..4624
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4484..4613
                     /label=AAV2 ITR
     rep_origin      4699..5154
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4786..4805)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4996..5017
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5171..5190)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5290..5312
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5350..5368)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5436..5540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5541..6401
                     /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(5759..5778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     5933..5954
                     /label=Amp-F1
     primer_bind     complement(5935..5957)
                     /label=Amp-R1
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg
      841 ctctaggaag atctaacata tcctggtgtg gagtagggga cgctgctctg acagaggctc
      901 gggggcctga gctggctctg tgagctgggg aggaggcaga cagccaggcc ttgtctgcaa
      961 gcagacctgg cagcattggg ctggccgccc cccagggcct cctcttcatg cccagtgaat
     1021 gactcacctt ggcacagaca caatgttcgg ggtgggcaca gtgcctgctt cccgccgcac
     1081 cccagccccc ctcaaatgcc ttccgagaag cccattgagc agggggcttg cattgcaccc
     1141 cagcctgaca gcctggcatc ttgggataaa agcagcacag ccccctaggg gctgcccttg
     1201 ctgtgtggcg ccaccggcgg tggagaacaa ggctctattc agcctgtgcc caggaaaggg
     1261 gatcagggga tgcccaggca tggacagtgg gtggcagggg gggagaggag ggctgtctgc
     1321 ttcccagaag tccaaggaca caaatgggtg aggggagagc tctccccata gctgggctgc
     1381 ggcccaaccc caccccctca ggctatgcca gggggtgttg ccaggggcac ccgggcatcg
     1441 ccagtctagc ccactccttc ataaagccct cgcatcccag gagcgagcag agccagagca
     1501 ggttggagag gagacgcatc acctccgctg ctcgcaagct ttattgcggt agtttatcac
     1561 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcaga
     1621 agttggtcgt gaggcactgg gcaggtaagt atcaaggtta caagacaggt ttaaggagac
     1681 caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt
     1741 ggtcttactg acatccactt tgcctttctc tccacaggtg tccactccca gttcaattac
     1801 agctcttaag gctagagtac ttaatacgac tcactatagg ctagcctcga gaattcgcca
     1861 ccatggccag cagcctgtac agcttcctgc tggccctgag catcgtgtac atcttcgtgg
     1921 cccccaccca cagctatcca tatgatgttc cagattatgc tggggcccag ccggccagat
     1981 ctatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2041 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2101 gccgccccta cgaggccttc cagaccgcta ggctgaaggt gaccaagggt ggccccctgc
     2161 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacattaagc
     2221 acccagccga catccccgac tacttcaagc tgtccttccc cgagggcttc aggtgggagc
     2281 gcgtgatgag cttcgaggac ggcggcatta ttcacgttaa tcaggactcc tccctgcagg
     2341 acggcgtatt catctacaag gtgaagctgc tcggcaccaa cttccccccc gacggccccg
     2401 taatgcagaa gaagaccatg ggctgggagg cttcccccgg catctgggac ttcgtgaaga
     2461 gcgccggcag gaagatcggc atcggcagcg aggaggccgc ccccgccgcc gacgacctga
     2521 agaaggagct ggacagctac aagctgggca ccgacgacgt gaaggtggag gtggtgggcg
     2581 acaaggccgt gatcaccggc aacgtggccg accagagcat cctggagaag gccatcgtgg
     2641 ccgtgggcaa caccctgggc atcagcaagg tggagagcaa cgtggaggtg cccgacgcca
     2701 aggagcccgt gttccacacc gtgaagaagg gcgacaccct gtgggccatc gccagcaagc
     2761 agtacggcaa cgtcagcaag tacaacgcca tcttcgaggc caacaagccc atgctgagcc
     2821 accccgacaa gatctacccc ggccaggtgc tgaggatccc cgacctgggc tctgcctgcg
     2881 tttccgagtg gatgtacccc gaggacggcg ccctgaagag cgagatcagg aaggggctga
     2941 ggctgaagga cggcggccac tacgccgccg aggtcaagac cacctacaag gccaagaagc
     3001 ccgtgcagct gcccggcgcc tacatcgtcg acatcgagtt ggacatcttg tcccacaacg
     3061 aggactacac catcgtggaa cagtgcgaac gcgccgtggg ccgccacccc accggtggca
     3121 cggacgcgct gtacaagggc gtcgacgaac aaaaactcat ctcagaagag gatctgaatg
     3181 ctgtgggcca ggacacgcag gaggtcatcg tggtgccaca ctccttgccc tttaaggtgg
     3241 tggtgatctc agccatcctg gccctggtgg tgctcaccat catctccctt atcatcctca
     3301 tcatgctttg gcagaagaag ccacgttagg atatcaagct tatcgataat caacctctgg
     3361 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3421 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3481 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3541 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3601 ccaccacctg tcagctcctt tccggaactt tcgctttccc cctccctatt gccacggcgg
     3661 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3721 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca
     3781 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3841 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3901 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga
     3961 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     4021 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     4081 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     4141 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     4201 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     4261 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     4321 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     4381 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     4441 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     4501 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4561 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4621 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4681 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4741 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4801 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4861 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4921 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4981 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5041 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5101 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5161 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5221 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5281 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5341 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5401 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5461 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5521 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5581 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5641 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5701 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5761 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5821 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5881 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5941 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6001 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6061 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6121 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6181 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6241 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6301 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6361 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6421 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6481 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6541 cagaccccgt agaaaagatc aaaggatctt c
//