LOCUS       sequence-412159-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the genetically-encoded fluorescent 
            corticotropin-releasing factor (CRF) sensor GRAB_CRF0.5 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_CRF0.5
FEATURES             Location/Qualifiers
     source          1..6884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     64..83
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   225..354
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(829..862)
                     /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(913..946)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1426..1445
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1685..1706
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1812..1833)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2993..3017)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(3021..3080)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      3077..3086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3093..3126
                     /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    3177..3210
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3235..3823
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3288..3308)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3706..3717)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3855..4331
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4054..4073)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4371..4511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4371..4500
                     /label=AAV2 ITR
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5177..5199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5237..5255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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(5646..5665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6459..6884,1..163)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
       61 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
      121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
      181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
      241 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
      301 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
      361 gcacgcgtat gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca
      421 agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca
      481 ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg ccttcgcccc
      541 cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg
      601 gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag
      661 ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg
      721 gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc
      781 tgagagcgca gtcgagaaac cggtgccacc tctagagtcg actccggaat aacttcgtat
      841 aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata
      901 tgaaacctct taataacttc gtatagcata cattatacga agttatggcg catatgttac
      961 aagactgctg tggactgctt gatgctgtga aagctgacac gggttgggga ggtggggatg
     1021 gacatggcac gggccactcg ggcacggatc gagtgcttgt cctgccaccg gtgccacctc
     1081 ttccggatgg cagaacggac ctcactattg aggaaacagt agaacacaga cacaaagaag
     1141 ccctggaagg attccaggaa ggagttgaag tagatgaaga cgacccggga gacctcatcc
     1201 tccccgggat tgacgaagaa cagcatgtag gtgatgccca ggaggggcag cagcaccaga
     1261 gtggctttca cagccttcct gtactgccgc ccgcgccagc gtgctgctgc tccggtgttg
     1321 tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg
     1381 atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg
     1441 tcgtccttga agaagatggt gcgctcctgg atgtagcctt cgggcatggc ggacttgaag
     1501 aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg
     1561 gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg
     1621 gtcagcttgc cctcggtggc atcgccctca ccctcgccgg acacgctgaa cttgtggccg
     1681 tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc
     1741 tttctcacca tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc gagagtgatc
     1801 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttc
     1861 gaaagtttgg actgcacgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     1921 atgggggtgt tctgctggta gtggtaggcg agctgcacgc cgccgtcctc gatgttgtgg
     1981 cggatgtgga agttcgcctt gatgccgttc ttctgcttgt cggccttgat atagacgttt
     2041 cctccctcct ccaggtccag cgcgtcggtg tcgcgcgggg tggatgcccg gagcttggtc
     2101 atgaggatgc ggacgatgtt gaaaaggaag atgaaattga tcagcaggac caggatcatg
     2161 gggccctggt agatgtagtc ggtgtacacc ccaggccttt tgccaaacca gcacttctca
     2221 ttgtcgtagt acagcttccc aatggcccag gccacaatga tggggaaggg cacaccccag
     2281 ccaatgcaga tgaacatcca tttgcgcagc cggtcagtgg agtaggtgag cacgatggct
     2341 gtgtgcaggt agcagccctc gccgaacatc cagaagaagt tggtcacatg gaagtagttg
     2401 taggcggctg tcaccaacct gcaccagccc acgttgctct ggtggacctc ggggctcatg
     2461 gttagctgga ccacgaacca ggtggcgttg cgcaggatga aggcggagat gaggttccag
     2521 tggatgatgt ttcgcaggca ccggatgctc ctgagccgca gaaagaggac aaaggccacc
     2581 aggagggcca ccagggagat acagtggccc aggtagttga tgatgactgc gacatggtag
     2641 tgcaccttgc tttttttctc ctcattgagg atctcctggc actcggagta attcacgcgg
     2701 gcggcccagc tgccattggc caggcactcc cggtagccat tgtttgtggt attgtagcgg
     2761 acaccataga aaaaggcagg gcagggccga accactagct gccccgcagg gctgcggggc
     2821 cagcaggtgc caatgaggtc cacggatgcg ttgcactgca gtcctgagat gttgctggcc
     2881 agggacaggc tctcgcagtg ctggtcctgg agggaggcag agacggggtt cagccccaga
     2941 aggagaaggg ccttgacgag acggagctgc gggtgccctc ccataccggt ggtgccaact
     3001 tttttgtaca aacttgtgtc accagtggaa cctggaaccc agagcagcag tacccatagc
     3061 aggagtgtgt ctgtctccat ggtggcgcta gcataacttc gtataaagta tcctatacga
     3121 agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa
     3181 cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat cgataatcaa
     3241 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3301 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3361 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3421 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3481 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3541 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3601 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat
     3661 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3721 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     3781 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg
     3841 ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     3901 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     3961 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     4021 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     4081 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     4141 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     4201 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     4261 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     4321 tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct
     4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4501 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaa
//
