LOCUS       sequence-412146-        6660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the genetically-encoded fluorescent 
            corticotropin-releasing factor (CRF) control sensor GRAB_CRFmut in a
            cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6660)
  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 6660)
  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-EFS-DIO-GRAB_CRFmut
FEATURES             Location/Qualifiers
     source          1..6660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..73
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   215..344
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        365..576
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    complement(595..628)
                     /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(679..712)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1192..1211
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1451..1472
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1578..1599)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2759..2783)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2787..2846)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      2843..2852
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2859..2892
                     /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    2943..2976
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3054..3074)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3472..3483)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3621..4097
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3820..3839)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4137..4277
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4137..4266
                     /label=AAV2 ITR
     rep_origin      4352..4807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4439..4458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4649..4670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4824..4843)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4943..4965
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5003..5021)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5089..5193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5194..5262,5263..6054)
                     /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(5412..5431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6225..6660,1..153)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
       61 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      121 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      181 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
      241 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
      301 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtaa
      361 gcttgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
      421 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      481 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      541 gttctttttc gcaacgggtt tgccgccaga acacagtcta gagtcgactc cggaataact
      601 tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta
      661 gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt atggcgcata
      721 tgttacaaga ctgctgtgga ctgcttgatg ctgtgaaagc tgacacgggt tggggaggtg
      781 gggatggaca tggcacgggc cactcgggca cggatcgagt gcttgtcctg ccaccggtgc
      841 cacctcttcc ggatggcaga acggacctca ctattgagga aacagtagaa cacagacaca
      901 aagaagccct ggaaggattc caggaaggag ttgaagtaga tgaagacgac ccgggagacc
      961 tcatcctccc cgggattgac gaagaacagc atgtaggtga tgcccaggag gggcagcagc
     1021 accagagtgg ctttcacagc cttcctcatc tgcctcccgc gccagcgtgc tgctgctccg
     1081 gtgttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     1141 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     1201 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt agccttcggg catggcggac
     1261 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag
     1321 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     1381 ttcagggtca gcttgccctc ggtggcatcg ccctcaccct cgccggacac gctgaacttg
     1441 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     1501 tcgccctttc tcaccatgct ccctccggta ccgcccttgt acagctcgtc catgccgaga
     1561 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     1621 tctttcgaaa gtttggactg cacgctcagg tagtggttgt cgggcagcag cacggggccg
     1681 tcgccgatgg gggtgttctg ctggtagtgg taggcgagct gcacgccgcc gtcctcgcag
     1741 ttgtggcgga tccagaagtt cgccttgatg ccgttcttct gcttgtcggc cttgatatag
     1801 acgtttcctc cctcctccag gtccagcgcg tcggtgtcgg ggccggtgta tgcccggagc
     1861 ttggtcatga ggatgcggac gatgttgaaa aggaagatga aattgatcag caggaccagg
     1921 atcctggggc cctggtagat gtagtcggtg tacaccccag gccttttgcc aaaccagcac
     1981 ttctcattgt cgtagtacag cttcccaatg gcccaggcca caatgatggg gaagggcaca
     2041 ccccagccaa tgcagatgaa catccatttg cgcagccggt cagtggagta ggtgagcacg
     2101 atggctgtgt gcaggtagca gccctcgccg aacatccaga agaagttggt cacatggaag
     2161 tagttgtagg cggctgtcac caacctgcac cagcccacgt tgctctggtg gacctcgggg
     2221 ctcatggtta gctggaccac gaaccaggtg gcgttgcgca ggatgaaggc ggagatgagg
     2281 ttccagtgga tgatgtttcg caggcaccgg atgctcctga gccgcagaaa gaggacaaag
     2341 gccaccagga gggccaccag ggagatacag tggcccaggt agttgatgat gactgcgaca
     2401 tggtagtgca ccttgctttt tttctcctca ttgaggatct cctggcactc ggagtaattc
     2461 acgcgggcgg cccagctgcc attggccagg cactcccggt agccattgtt tgtggtattg
     2521 tagcggacac catagaaaaa ggcagggcag ggccgaacca ctagctgccc cgcagggctg
     2581 cggggccagc aggtgccaat gaggtccacg gatgcgttgc actgcagtcc tgagatgttg
     2641 ctggccaggg acaggctctc gcagtgctgg tcctggaggg aggcagagac ggggttcagc
     2701 cccagaagga gaagggcctt gacgagacgg agctgcgggt gccctcccat accggtggtg
     2761 ccaacttttt tgtacaaact tgtgtcacca gtggaacctg gaacccagag cagcagtacc
     2821 catagcagga gtgtgtctgt ctccatggtg gcgctagcat aacttcgtat aaagtatcct
     2881 atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa
     2941 gaataacttc gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat
     3001 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3061 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3121 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3181 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3241 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3301 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3361 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3421 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3481 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3541 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     3601 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3661 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3721 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3781 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     3841 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     3901 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     3961 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     4021 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     4081 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     4141 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4201 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4261 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4321 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4381 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4441 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4501 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4561 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4621 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4681 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4741 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4801 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4861 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4921 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4981 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5041 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5101 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5161 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5221 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5281 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5341 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5401 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5461 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5521 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5581 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5641 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5701 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5761 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5821 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5881 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5941 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6001 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6061 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6361 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
//
