LOCUS Exported 7479 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Green/red FRET biosensor to monitor RhoA nucleotide loading state and activity. ACCESSION . VERSION . KEYWORDS pTriEx-RhoA-wt_mScarlet-i_SGFP2 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7479 /organism="synthetic DNA construct" /mol_type="other DNA" 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..1580 /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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 3317..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 4007..4585 /codon_start=1 /product="human Rho family small GTPase RhoA" /label=RhoA /translation="AAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADI EVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKH 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 5756..5789 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." 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(6557..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 gcgtccaccg 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 aggaagatta 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 //