pAAV-EFS-DIO-GRAB_CRF1.0

LOCUS       pAAV-EFS-DIO-GRAB_CRF1.0             6660 bp DNA     circular SYN 23-JAN-2024
DEFINITION  Expresses the genetically-encoded fluorescent 
            corticotropin-releasing factor (CRF) sensor GRAB_CRF1.0 in a 
            cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EFS-DIO-GRAB_CRF1.0
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 Jul 8, 2026 from SnapGene 6.0.2
            https://www.biofeng.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"
FEATURES             Location/Qualifiers
     source          1..6660
                     /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"
     promoter        375..586
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    605..638
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    689..722
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(733..2856)
                     /codon_start=1
                     /label=CRF1.0
                     /translation="METDTLLLWVLLLWVPGSTGDTSLYKKVGTTGMGGHPQLRLVKAL
                     LLLGLNPVSASLQDQHCESLSLASNISGLQCNASVDLIGTCWPRSPAGQLVVRPCPAFF
                     YGVRYNTTNNGYRECLANGSWAARVNYSECQEILNEEKKSKVHYHVAVIINYLGHCISL
                     VALLVAFVLFLRLRSIRCLRNIIHWNLISAFILRNATWFVVQLTMSPEVHQSNVGWCRL
                     VTAAYNYFHVTNFFWMFGEGCYLHTAIVLTYSTDRLRKWMFICIGWGVPFPIIVAWAIG
                     KLYYDNEKCWFGKRPGVYTDYIYQGPMILVLLINFIFLFNIVRILMTKLRAYTGPDTDA
                     LDLEEGGNVYIKADKQKNGIKANFTIRHNCEDGGVQLATHYQQNTPIGDGPVLLPDNHY
                     LSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPI
                     LVELDGDVNGHKFSVSGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR
                     YPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE
                     DGNILGHKLEYNTGAAARWRGRQMRKAVKATLVLLPLLGITYMLFFVNPGEDEVSRVVF
                     IYFNSFLESFQGFFVSVFYCFLNSEVRSAIRKRWHRWQDKHSIRARVARAMSIPTSPTR
                     VSFHSIKQSTAVL"
     primer_bind     1202..1221
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1461..1482
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1588..1609)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2769..2793)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(2794..2856)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     regulatory      2853..2862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2869..2902)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(2953..2986)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3011..3599
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3064..3084)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3482..3493)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3631..4107
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3830..3849)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4147..4287
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4147..4276
                     /label=AAV2 ITR
     rep_origin      4362..4817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4659..4680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4834..4853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4953..4975
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5013..5031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5099..5203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5204..6064
                     /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(5422..5441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6235..6660,1..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 gcacgcgtaa gcttgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      421 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      481 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      541 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagtcta gagtcgactc
      601 cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta
      661 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt
      721 atggcgcata tgttacaaga ctgctgtgga ctgcttgatg ctgtgaaagc tgacacgggt
      781 tggggaggtg gggatggaca tggcacgggc cactcgggca cggatcgagt gcttgtcctg
      841 ccaccggtgc cacctcttcc ggatggcaga acggacctca ctattgagga aacagtagaa
      901 cacagacaca aagaagccct ggaaggattc caggaaggag ttgaagtaga tgaagacgac
      961 ccgggagacc tcatcctccc cgggattgac gaagaacagc atgtaggtga tgcccaggag
     1021 gggcagcagc accagagtgg ctttcacagc cttcctcatc tgcctcccgc gccagcgtgc
     1081 tgctgctccg gtgttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     1141 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     1201 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt agccttcggg
     1261 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
     1321 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     1381 gcagatgaac ttcagggtca gcttgccctc ggtggcatcg ccctcaccct cgccggacac
     1441 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     1501 gaacagctcc tcgccctttc tcaccatgct ccctccggta ccgcccttgt acagctcgtc
     1561 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     1621 ctcgttgggg tctttcgaaa gtttggactg cacgctcagg tagtggttgt cgggcagcag
     1681 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg gtggcgagct gcacgccgcc
     1741 gtcctcgcag ttgtggcgga tggtgaagtt cgccttgatg ccgttcttct gcttgtcggc
     1801 cttgatatag acgtttcctc cctcctccag gtccagcgcg tcggtgtcgg ggccggtgta
     1861 tgcccggagc ttggtcatga ggatgcggac gatgttgaaa aggaagatga aattgatcag
     1921 caggaccagg atcatggggc cctggtagat gtagtcggtg tacaccccag gccttttgcc
     1981 aaaccagcac ttctcattgt cgtagtacag cttcccaatg gcccaggcca caatgatggg
     2041 gaagggcaca ccccagccaa tgcagatgaa catccatttg cgcagccggt cagtggagta
     2101 ggtgagcacg atggctgtgt gcaggtagca gccctcgccg aacatccaga agaagttggt
     2161 cacatggaag tagttgtagg cggctgtcac caacctgcac cagcccacgt tgctctggtg
     2221 gacctcgggg ctcatggtta gctggaccac gaaccaggtg gcgttgcgca ggatgaaggc
     2281 ggagatgagg ttccagtgga tgatgtttcg caggcaccgg atgctcctga gccgcagaaa
     2341 gaggacaaag gccaccagga gggccaccag ggagatacag tggcccaggt agttgatgat
     2401 gactgcgaca tggtagtgca ccttgctttt tttctcctca ttgaggatct cctggcactc
     2461 ggagtaattc acgcgggcgg cccagctgcc attggccagg cactcccggt agccattgtt
     2521 tgtggtattg tagcggacac catagaaaaa ggcagggcag ggccgaacca ctagctgccc
     2581 cgcagggctg cggggccagc aggtgccaat gaggtccacg gatgcgttgc actgcagtcc
     2641 tgagatgttg ctggccaggg acaggctctc gcagtgctgg tcctggaggg aggcagagac
     2701 ggggttcagc cccagaagga gaagggcctt gacgagacgg agctgcgggt gccctcccat
     2761 accggtggtg ccaacttttt tgtacaaact tgtgtcacca gtggaacctg gaacccagag
     2821 cagcagtacc catagcagga gtgtgtctgt ctccatggtg gcgctagcat aacttcgtat
     2881 aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga
     2941 atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat tcgatatcaa
     3001 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3061 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3121 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3181 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3241 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3301 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3361 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3421 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3481 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3541 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3601 tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
     3661 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     3721 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     3781 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     3841 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     3901 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     3961 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     4021 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     4081 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc
     4141 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4201 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4261 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4321 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4381 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4441 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4501 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4561 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4621 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4681 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4741 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4801 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4861 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4921 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4981 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5041 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5101 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5161 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5221 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5281 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5341 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5401 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5461 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5521 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5581 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5641 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5701 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5761 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5821 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5881 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5941 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6001 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6061 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6121 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6181 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6241 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6301 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6361 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6421 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6481 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6541 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6601 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
//