LOCUS       sequence-412144-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the genetically-encoded fluorescent 
            corticotropin-releasing factor (CRF) sensor GRAB_CRF1.0 in a 
            cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  AUTHORS   Wang H, Qian T, Zhao Y, Zhuo Y, Wu C, Osakada T, Chen P, Chen Z, Ren
            H, Yan Y, Geng L, Fu S, Mei L, Li G, Wu L, Jiang Y, Qian W, Zhang L,
            Peng W, Xu M, Hu J, Jiang M, Chen L, Tang C, Zhu Y, Lin D, Zhou JN, 
            Li Y
  TITLE     A tool kit of highly selective and sensitive genetically encoded 
            neuropeptide sensors.
  JOURNAL   Science. 2023 Nov 17;382(6672):eabq8173. doi: 
            10.1126/science.abq8173. Epub 2023 Nov 17.
  PUBMED    37972184
REFERENCE   2  (bases 1 to 6884)
  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: "2023-11-17"; volume: "382(6672):eabq8173. doi"; pages: " 
            10.1126/science.abq8173. Epub 2023 Nov 17"
COMMENT     Sequence Label: pAAV-hSyn-DIO-GRAB_CRF1.0
FEATURES             Location/Qualifiers
     source          1..6884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   212..341
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(816..849)
                     /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(900..933)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1413..1432
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1672..1693
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1799..1820)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2980..3004)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(3008..3067)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      3064..3073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3080..3113
                     /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    3164..3197
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3222..3810
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3275..3295)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3693..3704)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3842..4318
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4041..4060)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4358..4498
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4358..4487
                     /label=AAV2 ITR
     rep_origin      4573..5028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4660..4679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4870..4891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5045..5064)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5164..5186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5224..5242)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5310..5414
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5415..5483,5484..6275)
                     /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(5633..5652)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6446..6884,1..150)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
       61 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      181 ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc
      241 actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
      301 gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca cgcgtatgag
      361 gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc
      421 cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg
      481 cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc
      541 gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac
      601 tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca
      661 cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc
      721 agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc
      781 gagaaaccgg tgccacctct agagtcgact ccggaataac ttcgtatagg atactttata
      841 cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa
      901 taacttcgta tagcatacat tatacgaagt tatggcgcat atgttacaag actgctgtgg
      961 actgcttgat gctgtgaaag ctgacacggg ttggggaggt ggggatggac atggcacggg
     1021 ccactcgggc acggatcgag tgcttgtcct gccaccggtg ccacctcttc cggatggcag
     1081 aacggacctc actattgagg aaacagtaga acacagacac aaagaagccc tggaaggatt
     1141 ccaggaagga gttgaagtag atgaagacga cccgggagac ctcatcctcc ccgggattga
     1201 cgaagaacag catgtaggtg atgcccagga ggggcagcag caccagagtg gctttcacag
     1261 ccttcctcat ctgcctcccg cgccagcgtg ctgctgctcc ggtgttgtac tccagcttgt
     1321 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     1381 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     1441 agatggtgcg ctcctggatg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     1501 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     1561 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccct
     1621 cggtggcatc gccctcaccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     1681 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttt ctcaccatgc
     1741 tccctccggt accgcccttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     1801 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttcgaa agtttggact
     1861 gcacgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     1921 gctggtagtg ggtggcgagc tgcacgccgc cgtcctcgca gttgtggcgg atggtgaagt
     1981 tcgccttgat gccgttcttc tgcttgtcgg ccttgatata gacgtttcct ccctcctcca
     2041 ggtccagcgc gtcggtgtcg gggccggtgt atgcccggag cttggtcatg aggatgcgga
     2101 cgatgttgaa aaggaagatg aaattgatca gcaggaccag gatcatgggg ccctggtaga
     2161 tgtagtcggt gtacacccca ggccttttgc caaaccagca cttctcattg tcgtagtaca
     2221 gcttcccaat ggcccaggcc acaatgatgg ggaagggcac accccagcca atgcagatga
     2281 acatccattt gcgcagccgg tcagtggagt aggtgagcac gatggctgtg tgcaggtagc
     2341 agccctcgcc gaacatccag aagaagttgg tcacatggaa gtagttgtag gcggctgtca
     2401 ccaacctgca ccagcccacg ttgctctggt ggacctcggg gctcatggtt agctggacca
     2461 cgaaccaggt ggcgttgcgc aggatgaagg cggagatgag gttccagtgg atgatgtttc
     2521 gcaggcaccg gatgctcctg agccgcagaa agaggacaaa ggccaccagg agggccacca
     2581 gggagataca gtggcccagg tagttgatga tgactgcgac atggtagtgc accttgcttt
     2641 ttttctcctc attgaggatc tcctggcact cggagtaatt cacgcgggcg gcccagctgc
     2701 cattggccag gcactcccgg tagccattgt ttgtggtatt gtagcggaca ccatagaaaa
     2761 aggcagggca gggccgaacc actagctgcc ccgcagggct gcggggccag caggtgccaa
     2821 tgaggtccac ggatgcgttg cactgcagtc ctgagatgtt gctggccagg gacaggctct
     2881 cgcagtgctg gtcctggagg gaggcagaga cggggttcag ccccagaagg agaagggcct
     2941 tgacgagacg gagctgcggg tgccctccca taccggtggt gccaactttt ttgtacaaac
     3001 ttgtgtcacc agtggaacct ggaacccaga gcagcagtac ccatagcagg agtgtgtctg
     3061 tctccatggt ggcgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt
     3121 aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt
     3181 atgctatacg aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca
     3241 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3301 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3361 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3421 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3481 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3541 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3601 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     3661 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3721 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3781 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     3841 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     3901 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     3961 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     4021 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     4081 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     4141 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     4201 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     4261 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4321 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     4381 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4441 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4501 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4561 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4621 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4681 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4741 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4801 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4861 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4921 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4981 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5041 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5101 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5161 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5221 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5281 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5341 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5401 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5461 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5521 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5581 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5641 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5701 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5761 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5821 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5881 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5941 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6001 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6061 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6121 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6181 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6241 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6301 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6361 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6421 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6481 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6541 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6601 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6661 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6721 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6781 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6841 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtat
//
