LOCUS       sequence-340567-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Rac1 FRET sensor RaichuEV-Rac1 which contains a dominant
            negative mutation Y40C in the Rac1 domain. More useful than T17N 
            mutation due to less harmful to endogenous signaling..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  AUTHORS   Aihara S, Fujimoto S, Sakaguchi R, Imai T
  TITLE     BMPR-2 gates activity-dependent stabilization of primary dendrites 
            during mitral cell remodeling.
  JOURNAL   Cell Rep. 2021 Jun 22;35(12):109276. doi: 
            10.1016/j.celrep.2021.109276.
  PUBMED    34161760
REFERENCE   2  (bases 1 to 7378)
  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: "Cell Rep.";
            date: "2021-06-22"; volume: "35(12):109276. doi"; pages: " 
            10.1016/j.celrep.2021.109276"
COMMENT     Sequence Label: pCAGGS-RaichuEV-Rac1(Y40C)
FEATURES             Location/Qualifiers
     source          1..7378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1735..1737,1738..1740,1741..2448)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     CDS             2716..3063
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             join(3604..3606,3607..3609,3610..4122)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(4399..4418)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4464..4519
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4518..4537)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4880..4896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4880..4896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4893..4915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4904..4920
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4928..4934,4935..4952,4953..4958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4973..4994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5053..5248
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5099..5234
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5254..5388
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5291..5310)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5345..5364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5456..5473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5627..6215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5707..5726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6386..7177,7178..7246))
                     /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     7009..7028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7247..7351)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggsgcgcg ccrggggggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt cggcatggtg
     1741 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1801 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1861 ctgaccctga agcttctatg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1921 accaccctgg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1981 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2041 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2101 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2161 gagtacaact acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     2221 aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac
     2281 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2341 agctaccagt ccgccctgtt caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2401 gagttcctga ccgccgccgg gatcactgag ggcatgaacg agctgtacct cgagaaagag
     2461 aaagagcggc cagagatttc tctcccttca gattttgaac acacaattca tgtcggtttt
     2521 gatgctgtca caggggagtt tacgggaatg ccagagcagt gggcccgctt gcttcagaca
     2581 tcaaatatca ctaagtcgga gcagaagaaa aacccgcagg ctgttctgga tgtgttggag
     2641 ttttacaact cgaagaagac atccaacagc cagaaataca tgagctttac agataagtca
     2701 gcttccggag gaaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     2761 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt
     2821 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     2881 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     2941 ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc tggtggtagt
     3001 accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     3061 ggtaccatgc aggccatcaa gtgtgtggtg gtgggagacg gagctgtagg taaaacttgc
     3121 ctactgatca gttacacaac caatgcattt cctggagaat atatccctac tgtctttgac
     3181 aattgttctg ccaatgttat ggtagatgga aaaccggtga atctgggctt atgggataca
     3241 gctggacaag aagattatga cagattacgc cccctatcct atccgcaaac agatgtgttc
     3301 ttaatttgct tttcccttgt gagtcctgca tcatttgaaa atgtccgtgc aaagtggtat
     3361 cctgaggtgc ggcaccactg tcccaacact cccatcatcc tagtgggaac taaacttgat
     3421 cttagggatg ataaagacac gatcgagaaa ctgaaggaga agaagctgac tcccatcacc
     3481 tatccgcagg gtctagccat ggctaaggag attggtgctg taaaatacct ggagtgctcg
     3541 gcgctcacac agcgaggcct caagacagtg tttgacgaag cgatccgagc agtccgcggc
     3601 cgcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3661 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3721 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3781 accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc cgaccacatg
     3841 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3901 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3961 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     4021 cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga caagcagaag
     4081 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     4141 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     4201 cactacttga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     4261 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgggccgc
     4321 tctagaaaga tgagcaaaga tggtaaaaag aagaaaaaga agtcaaagac aaagtgtgta
     4381 attatgtaag tcgacggatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     4441 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     4501 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     4561 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     4621 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     4681 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     4741 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     4801 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc
     4861 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4921 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4981 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5041 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5101 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5161 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5221 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     5281 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5341 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     5401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5461 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5521 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5581 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5641 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5701 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5761 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5821 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5881 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5941 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6001 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6061 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6121 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6181 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6241 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6301 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6361 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6421 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6481 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6541 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6601 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6661 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6721 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6781 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6841 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6901 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6961 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7021 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7081 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7141 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7201 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7261 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7321 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgg
//
