LOCUS       sequence-177185-        7845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PI(DM): Photo-inhibitable/dark mutant C450A; dc1.FLARE: built based 
            on GTPase FLARE dual chain sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7845)
  AUTHORS   Dagliyan O, Tarnawski M, Chu PH, Shirvanyants D, Schlichting I, 
            Dokholyan NV, Hahn KM
  TITLE     Engineering extrinsic disorder to control protein activity in living
            cells.
  JOURNAL   Science. 2016 Dec 16;354(6318):1441-1444.
  PUBMED    27980211
REFERENCE   2  (bases 1 to 7845)
  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: "2016-12-16"; volume: "354(6318)"; pages: "1441-1444"
COMMENT     Sequence Label: PI(DM)-Rac1(WT)dc1.FLARE
FEATURES             Location/Qualifiers
     source          1..7845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(509..565)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(575..628)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     promoter        complement(1654..1763)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     protein_bind    1779..1803
                     /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)."
     primer_bind     complement(1803..1822)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1804..1822)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1864..1883)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(1945..1967)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(2170..2372)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2199..2219)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2374..2677
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_recomb     2745..3572
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             2805..3410
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     CDS             complement(3448..3572)
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             join(3815..3883,3884..4675)
                     /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(4033..4052)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4688..5275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5177..5196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5443..5476
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_recomb     5485..6190
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             5485..6073
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     3'UTR           5730..6102
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     terminator      complement(6203..6250)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6320..6339
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    6338..6393
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     6439..6458
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(6548..6571)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(7028..7456)
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NARFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFIGVQLDGTEHVRDAAEREGVMLIKKTAENIDEAAKEL"
     CDS             complement(join(7634..7845,1..499,500..502))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2010)"
                     /label=mTurquoise
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(7676..7697)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggatcttgaa gttggccttg atgccgttct tctgcttgtc ggcggtgata tagacgttgt
       61 cgctgatgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
      121 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
      181 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
      241 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca
      301 cgccccagga cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
      361 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
      421 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
      481 acagctcctc gcccttgctc acgctagcag gtccagggtt ctcctccacg tctccagcct
      541 gcttcagcag gctgaagtta gtagctccgc ttcctgggcc aggattctcc tcgacgtcac
      601 cgcatgttag cagacttcct ctgccctctc cactgccatt aattttctgg ctgttggatg
      661 tcttcttcga gttgtaaaac tccaacacat ccagaacagc ctgcgggttt ttcttctgct
      721 ccgacttagt gatatttgat gtctgaagca agcgggccca ctgctctggc attcccgtaa
      781 actcccctgt gacagcatca aaaccgacat gaattgtgtg ttcaaaatct gaagggagag
      841 aaatctctgg ccgctctttc tctttctttt tatttgtttt atctccaggt aaaatggatc
      901 cgagctcaga tctgagtccg gacttgtaca gctcgtccat gccctcggtg atgccggcgg
      961 cggtcaggaa ctccagcagc accatgtggt cccgcttctc gttggggtcc ttgaacaggg
     1021 cgctctggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     1081 tgttctgctg gtagtggtcg gccagctgca cgccgccgtc ctcgatgttg tgccggatct
     1141 tgaagttggc cttgatgccg ttcttctgct tgtcggcggt gatgtacacg ttgtggctgt
     1201 tgtagttgta ctccagcttg tggcccagga tgttgccgtc ctccttgaag tcgatgccct
     1261 tcagctcgat ccggttcacc agggtgtcgc cctcgaactt cacctcggcc cgggtcttgt
     1321 agttgccgtc gtccttgaag aagatggtcc gctcctgcac gtagccctcg ggcatggcgc
     1381 tcttgaagaa gtcgtgctgc ttcatgtggt cggggtaccg ggcgaagcac tgcacgccgt
     1441 agcccagggt ggtcaccagg gtgggccagg gcacgggcag cttgccggtg gtgcacagca
     1501 gcttcagggt cagcttgccg taggtggcgt cgccctcgcc ctcgccgctc acgctgaact
     1561 tgtggccgtt cacgtcgccg tccagctcca ccaggatggg caccacgccg gtgaacagct
     1621 cttcgccctt gctcaccatg gtatatctcc tttgattgta aataaaatgt aatttacagt
     1681 atagtatttt aattaatata caaatgattt gataataatt cttatttaac tataatatat
     1741 tgtgttgggt tgaattaaag gtcccggatt aactccgggg aattgttatc cgctcacaat
     1801 tcccctatag tgagtcgtat taatttcggt ccgatccaat tctttgccaa aatgatgaga
     1861 cagcacaata accagcacgt tgcccaggag ctgtaggaaa aagaagaagg catgaacatg
     1921 gttagcagag gctctagagc cgccggtcac acgccagaag ccgaaccccg ccctgccccg
     1981 tcccccccga aggcagccgt ccccccgcgg acagccccga gccgctcccc ccgcacaaag
     2041 ggcccgatac ttacctgccc agtgcctcac gaccaacttc tgcacgttaa gttcgagact
     2101 ggcgttagca atttaactgt gataaactac cgcaataaag catctagtga tctgacggtt
     2161 cactaaacga cgtctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     2221 caacggggcg gggttattac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     2281 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     2341 acgcccattg gtgtactgcc aaaaccgcat cagcatggta atagcgatga ctaatacgta
     2401 gatgtactgc caagtaggaa agtcccgtaa ggtcatgtac tgggcataat gccaggcggg
     2461 ccatttaccg tcattgacgt caataggggg cggacttggc atatgataca cttgatgtac
     2521 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     2581 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     2641 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     2701 aactaatgac cccgtaattg attactatta ataggccggc catcagcaac tatatattga
     2761 tagacatttc cagtttgtga tattagtttg tgcgtctcat tacaatggct gttattttta
     2821 acaacaaaca actgctcgca gacaatagta tagaaaaggg aggtgaactg tttttgttta
     2881 acggttcgta caacattttg gaaagttatg ttaatccggt gctgctaaaa aatggtgtaa
     2941 ttgaactaga agaagctgcg tactatgccg gcaacatatt gtacaaaacc gacgatccca
     3001 aattcattga ttatataaat ttaataatta aagcaacaca ctccgaagaa ctaccagaaa
     3061 atagcactgt tgtaaattac agaaaaacta tgcgcagcgg tactatacac cccattaaaa
     3121 aagacatata tatttatgac aacaaaaaat ttactctata cgatagatac atatatggat
     3181 acgataataa ctatgttaat ttttatgaag agaaaaatga aaaagagaag gaatacgaag
     3241 aagaagacga caaggcgtct agtttatgtg aaaataaaat tatattgtcg caaattaact
     3301 gtgaatcatt tgaaaatgat tttaaatatt acctcagcga ttataactac gcgttttcaa
     3361 ttatagataa tactacaaat gttcttgttg cgtttggttt gtatcgttaa taaaaaacaa
     3421 atttgacatt tataattgtt ttattattca ataattacaa ataggattga gacccttgca
     3481 gttgccagca aacggacaga gcttgtcgac agttttcatt ttttggcagt ttggtggttt
     3541 atacacatct tgggatttga ttaaaagatg catgacttca cccacacact tggagtcgtg
     3601 gttgtagaaa tgcgtcagag ccttgattaa acatcggttt tcacacttgg cacacgaaac
     3661 cacgtttgca atgtaacagt ttagtataaa cctcttcctg tacataccgg cgcgtttata
     3721 cacatcttgg gatttgatta aaagatgcag gaaacgcgcg gacatgagac aataaccctg
     3781 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3841 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3901 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3961 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4021 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4081 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4141 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4201 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4261 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4321 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4381 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4441 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4501 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4561 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4621 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaacaaag
     4681 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4741 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4801 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4861 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4921 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4981 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5041 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5101 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5161 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5221 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg cggagcctat ggaaaaacgc
     5281 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5341 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5401 cgctcgccgc agccgaacga ccgagcgcag cgttccgagc ttataacttc gtataatgta
     5461 tgctatacga agttatgcga tcgcaattcc aaaaagctca actacaaatt tgatcgcgga
     5521 cgtgttagcc gacacaatta ataggcgtcg tgtggctatg gcaaaatcgt cttcggaagc
     5581 aacttctaac gacgagggtt gggacgacga cgataatcgg cctaataaag ctaacacgcc
     5641 cgatgttaaa tatgtccaag ctctatttaa cgtgtttacg tcgagtcaat tgtacactaa
     5701 cgacagtgat gaaagaaata caaaagcgca taatattttg aacgacgtcg aacctttatt
     5761 acaaaacaaa acacaaacga atatcgacaa agctagattg ctgctacaag atttggcaag
     5821 ttttgtggcg ttgagcgaaa atccattaga tagtccagcc atcggttcgg aaaaacaacc
     5881 cttgtttgaa actaatcgaa acctatttta caaatctatt gaggatttaa tatttaaatt
     5941 cagatataaa gacgctgaaa atcatttgat tttcgctcta acataccacc ctaaagatta
     6001 taaatttaat gaattattaa aatacgtaca acaattgtct gtaaatcaac aacgcacaga
     6061 atctagcgct taataaatgt actaataaca atgtatcgtg ttttaatacg ccggaccagt
     6121 gaacagaggt gcgtctggtg caaactcctt tactttgaac accagggaaa cttcaaggag
     6181 aatttcctcc gcatgctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     6241 gttatgctag ttacatatgg gcatatgttg ccaaactcta aaccaaatac tcattctgat
     6301 gttttaaatg atttgccctc ccatatgtcc ttccgagtga gagacacaaa aaattccaac
     6361 acactattgc aatgaaaata aatttccttt attagccaga agtcagatgc tcaaggggct
     6421 tcatgatgtc cccataattt ttggcagagg gaaaaagatc gatctcagtg gtatttgtga
     6481 gccagggcat tggccacacc agccaccacc ttctgatagg cagcctgcac ctgaggttaa
     6541 tcacttagtg atggtgatgg tgatggtggt gctcgagtta ctacaacagc aggcattttc
     6601 tcttcctctt cttcacggga ggcgggcaga ggactgctcg gatcgcttcg tcaaacactg
     6661 tcttgaggcc tcgctgtgtg agcgccgagc actccaggta ttttacggca ccaatctcct
     6721 tagccatggc tagaccctgc ggataggtga tgggagtcag cttcttctcc ttcagtttct
     6781 cgatcgtgtc tttatcatcc ctaagatcaa gtttagttcc cactaggatg atgggagtgt
     6841 tgggacagtg gtgccgcacc tcaggatacc actttgcacg gacattttca aatgatgcag
     6901 gactcacaag ggaaaagcaa attaagaaca catctgtttg cggataggat agggggcgta
     6961 atctgtcata atcttcttgt ccagctgtat cccataagcc cagattcacc ggttttccac
     7021 cactgccaag ttcttttgcc gcctcatcaa tattttctgc agttttctta atcagcatga
     7081 ctccctctct ctcggcagca tctcggacat gctcagttcc atccaactga accccaataa
     7141 agtactggac atctcccttc tgatctcgca taggctgcaa gtgaaagagg ttccagaact
     7201 ttttaccact ctttgtataa ttaatcagct gaacagtgac ctctgtttgg ttatctatgg
     7261 catctctaat ttttctcact gtcgcgcgat cagtttcagg accttgtaga aacctggcgt
     7321 ttcttcccaa aatttcttca cggctatatt ctgtcaactg caagaaacta tcggacgcga
     7381 atataatggg attatctggc aatcttgggt cagtaatgac aaagttcttc tcaatacgtt
     7441 caagtgtagt agccaaacct gatccatcta ccataacatt ggcagaataa ttgtcaaaga
     7501 cagtagggat atattctcca ggaaatgcat tggttgtgta actgatcagt aggcaagttt
     7561 tacctacagc tccgtctccc accaccacac acttgatggc ctgcatggat ccgagctcag
     7621 atctgagtcc ggacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     7681 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagt gcggactggg
     7741 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     7801 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggc
//
