LOCUS       sequence-177184-        7845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PI(LM): Photo-inhibitable/lit mutant I510E/I539E 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(LM)-Rac1(WT)dc1.FLARE
FEATURES             Location/Qualifiers
     source          1..7845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(192..394)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(221..241)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        396..699
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_recomb     767..1594
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             827..1432
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     CDS             complement(1470..1594)
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             join(1837..1905,1906..2697)
                     /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(2055..2074)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2710..3297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3199..3218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3465..3498
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_recomb     3507..4212
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             3507..4095
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     3'UTR           3752..4124
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     terminator      complement(4225..4272)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4342..4361
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    4360..4415
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     4461..4480
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(4570..4593)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(5050..5478)
                     /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
                     NCRFLQGPETDRAIVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFEGVQLDGTEHVRDAAEREGVMLIKKTAENEDEAAKEL"
     CDS             complement(join(5656..6366,6367..6369))
                     /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(5698..5719)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(6376..6432)
                     /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(6442..6495)
                     /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(7521..7630)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     protein_bind    7646..7670
                     /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(7670..7689)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7671..7689)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7731..7750)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(7812..7834)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cgtccccccc gaaggcagcc gtccccccgc ggacagcccc gagccgctcc ccccgcacaa
       61 agggcccgat acttacctgc ccagtgcctc acgaccaact tctgcacgtt aagttcgaga
      121 ctggcgttag caatttaact gtgataaact accgcaataa agcatctagt gatctgacgg
      181 ttcactaaac gacgtctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc
      241 gtcaacgggg cggggttatt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
      301 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat
      361 ccacgcccat tggtgtactg ccaaaaccgc atcagcatgg taatagcgat gactaatacg
      421 tagatgtact gccaagtagg aaagtcccgt aaggtcatgt actgggcata atgccaggcg
      481 ggccatttac cgtcattgac gtcaataggg ggcggacttg gcatatgata cacttgatgt
      541 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct
      601 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
      661 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
      721 tgaactaatg accccgtaat tgattactat taataggccg gccatcagca actatatatt
      781 gatagacatt tccagtttgt gatattagtt tgtgcgtctc attacaatgg ctgttatttt
      841 taacaacaaa caactgctcg cagacaatag tatagaaaag ggaggtgaac tgtttttgtt
      901 taacggttcg tacaacattt tggaaagtta tgttaatccg gtgctgctaa aaaatggtgt
      961 aattgaacta gaagaagctg cgtactatgc cggcaacata ttgtacaaaa ccgacgatcc
     1021 caaattcatt gattatataa atttaataat taaagcaaca cactccgaag aactaccaga
     1081 aaatagcact gttgtaaatt acagaaaaac tatgcgcagc ggtactatac accccattaa
     1141 aaaagacata tatatttatg acaacaaaaa atttactcta tacgatagat acatatatgg
     1201 atacgataat aactatgtta atttttatga agagaaaaat gaaaaagaga aggaatacga
     1261 agaagaagac gacaaggcgt ctagtttatg tgaaaataaa attatattgt cgcaaattaa
     1321 ctgtgaatca tttgaaaatg attttaaata ttacctcagc gattataact acgcgttttc
     1381 aattatagat aatactacaa atgttcttgt tgcgtttggt ttgtatcgtt aataaaaaac
     1441 aaatttgaca tttataattg ttttattatt caataattac aaataggatt gagacccttg
     1501 cagttgccag caaacggaca gagcttgtcg acagttttca ttttttggca gtttggtggt
     1561 ttatacacat cttgggattt gattaaaaga tgcatgactt cacccacaca cttggagtcg
     1621 tggttgtaga aatgcgtcag agccttgatt aaacatcggt tttcacactt ggcacacgaa
     1681 accacgtttg caatgtaaca gtttagtata aacctcttcc tgtacatacc ggcgcgttta
     1741 tacacatctt gggatttgat taaaagatgc aggaaacgcg cggacatgag acaataaccc
     1801 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     1861 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     1921 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     1981 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2041 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     2101 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     2161 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     2221 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     2281 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     2341 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     2401 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     2461 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     2521 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     2581 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     2641 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaacaa
     2701 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2761 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2821 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2881 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2941 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3001 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3061 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3121 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3181 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3241 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg ggcggagcct atggaaaaac
     3301 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3361 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3421 accgctcgcc gcagccgaac gaccgagcgc agcgttccga gcttataact tcgtataatg
     3481 tatgctatac gaagttatgc gatcgcaatt ccaaaaagct caactacaaa tttgatcgcg
     3541 gacgtgttag ccgacacaat taataggcgt cgtgtggcta tggcaaaatc gtcttcggaa
     3601 gcaacttcta acgacgaggg ttgggacgac gacgataatc ggcctaataa agctaacacg
     3661 cccgatgtta aatatgtcca agctctattt aacgtgttta cgtcgagtca attgtacact
     3721 aacgacagtg atgaaagaaa tacaaaagcg cataatattt tgaacgacgt cgaaccttta
     3781 ttacaaaaca aaacacaaac gaatatcgac aaagctagat tgctgctaca agatttggca
     3841 agttttgtgg cgttgagcga aaatccatta gatagtccag ccatcggttc ggaaaaacaa
     3901 cccttgtttg aaactaatcg aaacctattt tacaaatcta ttgaggattt aatatttaaa
     3961 ttcagatata aagacgctga aaatcatttg attttcgctc taacatacca ccctaaagat
     4021 tataaattta atgaattatt aaaatacgta caacaattgt ctgtaaatca acaacgcaca
     4081 gaatctagcg cttaataaat gtactaataa caatgtatcg tgttttaata cgccggacca
     4141 gtgaacagag gtgcgtctgg tgcaaactcc tttactttga acaccaggga aacttcaagg
     4201 agaatttcct ccgcatgctt tcagcaaaaa acccctcaag acccgtttag aggccccaag
     4261 gggttatgct agttacatat gggcatatgt tgccaaactc taaaccaaat actcattctg
     4321 atgttttaaa tgatttgccc tcccatatgt ccttccgagt gagagacaca aaaaattcca
     4381 acacactatt gcaatgaaaa taaatttcct ttattagcca gaagtcagat gctcaagggg
     4441 cttcatgatg tccccataat ttttggcaga gggaaaaaga tcgatctcag tggtatttgt
     4501 gagccagggc attggccaca ccagccacca ccttctgata ggcagcctgc acctgaggtt
     4561 aatcacttag tgatggtgat ggtgatggtg gtgctcgagt tactacaaca gcaggcattt
     4621 tctcttcctc ttcttcacgg gaggcgggca gaggactgct cggatcgctt cgtcaaacac
     4681 tgtcttgagg cctcgctgtg tgagcgccga gcactccagg tattttacgg caccaatctc
     4741 cttagccatg gctagaccct gcggataggt gatgggagtc agcttcttct ccttcagttt
     4801 ctcgatcgtg tctttatcat ccctaagatc aagtttagtt cccactagga tgatgggagt
     4861 gttgggacag tggtgccgca cctcaggata ccactttgca cggacatttt caaatgatgc
     4921 aggactcaca agggaaaagc aaattaagaa cacatctgtt tgcggatagg atagggggcg
     4981 taatctgtca taatcttctt gtccagctgt atcccataag cccagattca ccggttttcc
     5041 accactgcca agttcttttg ccgcctcatc ttcattttct gcagttttct taatcagcat
     5101 gactccctct ctctcggcag catctcggac atgctcagtt ccatccaact gaaccccttc
     5161 aaagtactgg acatctccct tctgatctcg cataggctgc aagtgaaaga ggttccagaa
     5221 ctttttacca ctctttgtat aattaatcag ctgaacagtg acctctgttt ggttatctat
     5281 ggcatctcta atttttctca ctatcgcgcg atcagtttca ggaccttgta gaaacctgca
     5341 gtttcttccc aaaatttctt cacggctata ttctgtcaac tgcaagaaac tatcggacgc
     5401 gaatataatg ggattatctg gcaatcttgg gtcagtaatg acaaagttct tctcaatacg
     5461 ttcaagtgta gtagccaaac ctgatccatc taccataaca ttggcagaat aattgtcaaa
     5521 gacagtaggg atatattctc caggaaatgc attggttgtg taactgatca gtaggcaagt
     5581 tttacctaca gctccgtctc ccaccaccac acacttgatg gcctgcatgg atccgagctc
     5641 agatctgagt ccggacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     5701 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gtgcggactg
     5761 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     5821 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     5881 ggccttgatg ccgttcttct gcttgtcggc ggtgatatag acgttgtcgc tgatgtagtt
     5941 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     6001 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     6061 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     6121 gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag cactgcacgc cccaggacag
     6181 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     6241 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     6301 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     6361 cttgctcacg ctagcaggtc cagggttctc ctccacgtct ccagcctgct tcagcaggct
     6421 gaagttagta gctccgcttc ctgggccagg attctcctcg acgtcaccgc atgttagcag
     6481 acttcctctg ccctctccac tgccattaat tttctggctg ttggatgtct tcttcgagtt
     6541 gtaaaactcc aacacatcca gaacagcctg cgggtttttc ttctgctccg acttagtgat
     6601 atttgatgtc tgaagcaagc gggcccactg ctctggcatt cccgtaaact cccctgtgac
     6661 agcatcaaaa ccgacatgaa ttgtgtgttc aaaatctgaa gggagagaaa tctctggccg
     6721 ctctttctct ttctttttat ttgttttatc tccaggtaaa atggatccga gctcagatct
     6781 gagtccggac ttgtacagct cgtccatgcc ctcggtgatg ccggcggcgg tcaggaactc
     6841 cagcagcacc atgtggtccc gcttctcgtt ggggtccttg aacagggcgc tctggtagct
     6901 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
     6961 gtggtcggcc agctgcacgc cgccgtcctc gatgttgtgc cggatcttga agttggcctt
     7021 gatgccgttc ttctgcttgt cggcggtgat gtacacgttg tggctgttgt agttgtactc
     7081 cagcttgtgg cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatccg
     7141 gttcaccagg gtgtcgccct cgaacttcac ctcggcccgg gtcttgtagt tgccgtcgtc
     7201 cttgaagaag atggtccgct cctgcacgta gccctcgggc atggcgctct tgaagaagtc
     7261 gtgctgcttc atgtggtcgg ggtaccgggc gaagcactgc acgccgtagc ccagggtggt
     7321 caccagggtg ggccagggca cgggcagctt gccggtggtg cacagcagct tcagggtcag
     7381 cttgccgtag gtggcgtcgc cctcgccctc gccgctcacg ctgaacttgt ggccgttcac
     7441 gtcgccgtcc agctccacca ggatgggcac cacgccggtg aacagctctt cgcccttgct
     7501 caccatggta tatctccttt gattgtaaat aaaatgtaat ttacagtata gtattttaat
     7561 taatatacaa atgatttgat aataattctt atttaactat aatatattgt gttgggttga
     7621 attaaaggtc ccggattaac tccggggaat tgttatccgc tcacaattcc cctatagtga
     7681 gtcgtattaa tttcggtccg atccaattct ttgccaaaat gatgagacag cacaataacc
     7741 agcacgttgc ccaggagctg taggaaaaag aagaaggcat gaacatggtt agcagaggct
     7801 ctagagccgc cggtcacacg ccagaagccg aaccccgccc tgccc
//
