LOCUS       sequence-163878-        7479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Green/red FRET biosensor for RhoA in the GTP locked constitutively 
            active state.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7479)
  AUTHORS   Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M,
            Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW Jr
  TITLE     mScarlet: a bright monomeric red fluorescent protein for cellular 
            imaging.
  JOURNAL   Nat Methods. 2016 Nov 21. doi: 10.1038/nmeth.4074.
  PUBMED    27869816
REFERENCE   2  (bases 1 to 7479)
  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: "Nat 
            Methods. 2016 Nov 21. doi: 10.1038/nmeth.4074."
COMMENT     Sequence Label: pTriEx-RhoA-Q63L_mScarlet-i_SGFP2
FEATURES             Location/Qualifiers
     source          1..7479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1931..1950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1931..1949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1950..1974
                     /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        1990..2099
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2120..2137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2420..2429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2426..3121
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2559..2577)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2827..2846
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(3317..3319,3320..3832)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIE"
     primer_bind     complement(3359..3380)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3620..3639)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3967..3988
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(4007..4189,4190..4192,4193..4585)
                     /codon_start=1
                     /product="constitutively active mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (Q63L)
                     /translation="AAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADI
                     EVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKH
                     FCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTK
                     DGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             4622..4654
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4661..4684
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4774..4793)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4839..4894
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4893..4912)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4982..5029
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5042..5747
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5130..5502
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5159..5747)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5756..5789)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(5957..6544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6036..6055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6557..7348,7349..7417))
                     /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     7180..7199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccggag
     1981 ttaatccggg acctttaatt caacccaaca caatatatta tagttaaata agaattatta
     2041 tcaaatcatt tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca
     2101 aaggagatat accatggcac accatcacca ccatcacgga tctggttcta gcctgggccc
     2161 cgtagagctg ctgctgcggg gctcctcgcg ccgcctcgac ctgctgcacc agcagctgca
     2221 ggagctgcac gcccacggtt ctggttccct gctagagcag ctgggcctgg ccggggcaga
     2281 cctggcggcc cccggggtac agcagcagct ggagctggag cgggagcggc tgcggcggga
     2341 aatccgcaag gagctgaagc tgaaggaggg tgctgagaac ctgcggcggg ccaccactga
     2401 cctgggccgc ggtaccggtg ccaccatggt gagcaagggc gaggcagtga tcaaggagtt
     2461 catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg
     2521 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2581 tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc
     2641 cttcatcaag caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt
     2701 caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac
     2761 ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc
     2821 tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcatccaccg agcggttgta
     2881 ccccgaggac ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg
     2941 ccgctacctg gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg
     3001 cgcctacaac gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt
     3061 ggaacagtac gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     3121 gtccggactc agatctaaca tcgaggaagc acaaaagcag aaggaacaga gacaggcagc
     3181 agaggagctg gcaaacggat ctgaagcaca aaagcagaag gaacagagac aggcagcaga
     3241 ggagctggca aacggatctg aagcacaaaa gcagaaggaa cagagacagg cagcagagga
     3301 gctggcaaac ggatccgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3361 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3421 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3481 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttcg cccgctaccc
     3541 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3601 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3661 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3721 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcaccgccga
     3781 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg
     3841 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3901 gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg
     3961 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg gctagcgctg ccatccggaa
     4021 gaaactggtg attgttggtg atggagcctg tggaaagaca tgcttgctca tagtcttcag
     4081 caaggaccag ttcccagagg tgtatgtgcc cacagtgttt gagaactatg tggcagatat
     4141 cgaggtggat ggaaagcagg tagagttggc tttgtgggac acagctgggc tggaagatta
     4201 tgatcgcctg aggcccctct cctacccaga taccgatgtt atactgatgt gtttttccat
     4261 cgacagccct gatagtttag aaaacatccc agaaaagtgg accccagaag tcaagcattt
     4321 ctgtcccaac gtgcccatca tcctggttgg gaataagaag gatcttcgga atgatgagca
     4381 cacaaggcgg gagcttgcca agatgaagca ggagccggtg aaacctgaag aaggcagaga
     4441 tatggcaaac aggattggcg cttttgggta catggagtgt tcagcaaaga ccaaagatgg
     4501 agtgagagag gtttttgaaa tggctacgag agctgctctg caagctagac gtgggaagaa
     4561 aaaatctggg tgccttgtct tgtgaaagct tgcggccgca cagctgtata cacgtgcaag
     4621 ccagccagaa ctcgccccgg aagaccccga ggatctcgag caccaccatc accatcacca
     4681 tcactaagtg attaacctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     4741 aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca aaaattatgg
     4801 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     4861 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     4921 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgta
     4981 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaagcatg
     5041 cggaggaaat tctccttgaa gtttccctgg tgttcaaagt aaaggagttt gcaccagacg
     5101 cacctctgtt cactggtccg gcgtattaaa acacgataca ttgttattag tacatttatt
     5161 aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat tttaataatt
     5221 cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt
     5281 ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg tttcgattag
     5341 tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga ttttcgctca
     5401 acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg
     5461 ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt
     5521 catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataga gcttggacat
     5581 atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc caaccctcgt
     5641 cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta ttaattgtgt
     5701 cggctaacac gtccgcgatc aaatttgtag ttgagctttt tggaattgcg atcgcataac
     5761 ttcgtatagc atacattata cgaagttata agctcggaac gctgcgctcg gtcgttcggc
     5821 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5881 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5941 ccgcgttgct ggcgtttttc cataggctcc gccccctgac gagcatcaca aaaatcgacg
     6001 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6061 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6121 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     6181 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6241 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6301 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6361 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6421 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6481 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6541 tcaagaagat cctttgttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6601 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6661 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6721 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6781 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6841 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6901 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6961 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7021 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7081 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7141 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7201 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7261 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7321 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7381 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7441 gaagcattta tcagggttat tgtctcatgt ccgcgcgtt
//
