LOCUS Exported 7518 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Second generation Rac1 FRET biosensor. ACCESSION . VERSION . KEYWORDS pTriEx4-Rac1-2G SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7518) AUTHORS Fritz RD, Menshykau D, Martin K, Reimann A, Pontelli V, Pertz O TITLE SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion. JOURNAL Dev Cell. 2015 Sep 29. pii: S1534-5807(15)00582-1. doi: 10.1016/j.devcel.2015.09.002. PUBMED 26439400 REFERENCE 2 (bases 1 to 7518) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7518 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 31..334 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 336..535 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 489..509 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 741..763 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 825..844 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" primer_bind 886..905 /label=T7 /note="T7 promoter, forward primer" promoter 886..904 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 905..929 /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 945..1054 /label=p10 promoter /note="baculovirus promoter for expression in insect cells" CDS 1075..1092 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1102..1131 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2377..2895 /codon_start=1 /product="N-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VN173 /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIE" primer_bind complement(2422..2443) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2683..2702) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3030..3051 /label=EGFP-C /note="EGFP, forward primer" CDS 3070..3648 /codon_start=1 /product="human Rac family small GTPase 1" /label=Rac1 /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVM VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR GLKTVFDEAIRAVLCPPPVKKRKRKCLLL" CDS 3655..3678 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" primer_bind complement(3768..3787) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 3833..3888 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(3887..3906) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 3976..4023 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_recomb 4036..4741 /label=baculovirus recombination region (ORF1629) /note="contains part of ORF1629" 3'UTR 4124..4496 /label=polyhedrin 3' UTR /note="3' UTR from the Autographa californica nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)" CDS complement(4153..4741) /codon_start=2 /product="baculovirus capsid-associated protein" /label=ORF1629 /note="required for viral replication" /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA" protein_bind 4750..4783 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." rep_origin complement(4951..5538) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5030..5049) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5551..6411) /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 6174..6193 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" misc_recomb 6654..7481 /label=baculovirus recombination region (lef2/ORF603) /note="contains ORF603 and part of lef2" CDS 6654..6778 /codon_start=3 /product="baculovirus late expression factor 2" /label=lef2 /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY" CDS complement(6816..7421) /codon_start=1 /product="baculovirus ORF603 protein" /label=ORF603 /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR" ORIGIN 1 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 61 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 121 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 181 cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg 241 taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca 301 gtacatctac gtattagtca tcgctattac catgctgatg cggttttggc agtacaccaa 361 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 421 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta ataaccccgc 481 cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagacgtcg 541 tttagtgaac cgtcagatca ctagatgctt tattgcggta gtttatcaca gttaaattgc 601 taacgccagt ctcgaactta acgtgcagaa gttggtcgtg aggcactggg caggtaagta 661 tcgggccctt tgtgcggggg gagcggctcg gggctgtccg cggggggacg gctgccttcg 721 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc 781 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat 841 tgtgctgtct catcattttg gcaaagaatt ggatcggacc gaaattaata cgactcacta 901 taggggaatt gtgagcggat aacaattccc cggagttaat ccgggacctt taattcaacc 961 caacacaata tattatagtt aaataagaat tattatcaaa tcatttgtat attaattaaa 1021 atactatact gtaaattaca ttttatttac aatcaaagga gatataccat ggcacaccat 1081 caccaccatc acgggagcgg ggagcagaaa ctaatatcag aggaggatct tggctcagga 1141 tcctccaccg acggcatgga cgagctgtac aagggtgcta gcggaggaat ggtgagcaag 1201 ggcgaggaga ccacaatggg cgtaatcaag cccgacatga agatcaagct gaagatggag 1261 ggcaacgtga atggccacgc cttcgtgatc gagggcgagg gcgagggcaa gccctacgac 1321 ggcaccaaca ccatcaacct ggaggtgaag gagggagccc ccctgccctt ctcctacgac 1381 attctgacca ccgcgttcgc ctacggcaac agggccttca ccaagtaccc cgacgacatc 1441 cccaactact tcaagcagtc cttccccgag ggctactctt gggagcgcac catgaccttc 1501 gaggacaagg gcatcgtgaa ggtgaagtcc gacatctcca tggaggagga ctccttcatc 1561 tacgagatac acctcaaggg cgagaacttc ccccccaacg gccccgtgat gcagaagaag 1621 accaccggct gggacgcctc caccgagagg atgtacgtgc gcgacggcgt gctgaagggc 1681 gacgtcaagc acaagctgct gctggagggc ggcggccacc accgcgttga cttcaagacc 1741 atctacaggg ccaagaaggc ggtgaagctg cccgactatc actttgtgga ccaccgcatc 1801 gagatcctga accacgacaa ggactacaac aaggtgaccg tttacgagag cgccgtggcc 1861 cgcaactccg gaaaagagaa agagcggcca gagatttctc tcccttcaga ttttgaacac 1921 acaattcatg tcggttttga tgctgtcaca ggggagttta cgggaatgcc agagcagtgg 1981 gcccgcttgc ttcagacatc aaatatcact aagtcggagc agaagaaaaa cccgcaggct 2041 gttctggatg tgttggagtt ttacaactcg aagaagacat ccaacagcca gaaatacatg 2101 agctttacag ataagtcagc tcccgggact tctggttctg gtaaacctgg ttctggtgaa 2161 ggttctacta aagggacttc tggttctggt aaacctggtt ctggtgaagg ttctactaaa 2221 gggacttctg gttctggtaa acctggttct ggtgaaggtt ctactaaagg gacttctggt 2281 tctggtaaac ctggttctgg tgaaggttct actaaagggg cggccgccat gatcactctc 2341 ggcatggacg agctgtacaa gggtggcagc ggtggcatgg tgagcaaggg cgaggagctg 2401 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2461 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc 2521 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc 2581 ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2641 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2701 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2761 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2821 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc 2881 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc 2941 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg 3001 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3061 gggggtacca tgcaggccat caagtgtgtg gtggtgggag acggagctgt aggtaaaact 3121 tgcctactga tcagttacac aaccaatgca tttcctggag aatatatccc tactgtcttt 3181 gacaattatt ctgccaatgt tatggtagat ggaaaaccgg tgaatctggg cttatgggat 3241 acagctggac aagaagatta tgacagatta cgccccctat cctatccgca aacagatgtg 3301 ttcttaattt gcttttccct tgtgagtcct gcatcatttg aaaatgtccg tgcaaagtgg 3361 tatcctgagg tgcggcacca ctgtcccaac actcccatca tcctagtggg aactaaactt 3421 gatcttaggg atgataaaga cacgatcgag aaactgaagg agaagaagct gactcccatc 3481 acctatccgc agggtctagc tatggccaag gagattggtg ctgtaaaata cctggagtgc 3541 tcggcgctca cacagcgagg cctcaagaca gtgtttgacg aagcgatccg agcagtcctc 3601 tgcccgcctc ccgtgaagaa gaggaagaga aaatgcctgc tgttgtgact cgagcaccac 3661 catcaccatc accatcacta agtgattaac ctcaggtgca ggctgcctat cagaaggtgg 3721 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct ttttccctct 3781 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg 3841 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca 3901 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca 3961 tatgcccata tgtaactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 4021 ttgctgaaag catgcggagg aaattctcct tgaagtttcc ctggtgttca aagtaaagga 4081 gtttgcacca gacgcacctc tgttcactgg tccggcgtat taaaacacga tacattgtta 4141 ttagtacatt tattaagcgc tagattctgt gcgttgttga tttacagaca attgttgtac 4201 gtattttaat aattcattaa atttataatc tttagggtgg tatgttagag cgaaaatcaa 4261 atgattttca gcgtctttat atctgaattt aaatattaaa tcctcaatag atttgtaaaa 4321 taggtttcga ttagtttcaa acaagggttg tttttccgaa ccgatggctg gactatctaa 4381 tggattttcg ctcaacgcca caaaacttgc caaatcttgt agcagcaatc tagctttgtc 4441 gatattcgtt tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa tattatgcgc 4501 ttttgtattt ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa acacgttaaa 4561 tagagcttgg acatatttaa catcgggcgt gttagcttta ttaggccgat tatcgtcgtc 4621 gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat tttgccatag ccacacgacg 4681 cctattaatt gtgtcggcta acacgtccgc gatcaaattt gtagttgagc tttttggaat 4741 tgcgatcgca taacttcgta tagcatacat tatacgaagt tataagctcg gaacgctgcg 4801 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 4861 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 4921 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc tgacgagcat 4981 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 5041 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 5101 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 5161 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5221 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5281 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5341 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 5401 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5461 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5521 agaaaaaaag gatctcaaga agatcctttg ttaccaatgc ttaatcagtg aggcacctat 5581 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5641 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5701 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5761 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5821 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5881 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 5941 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6001 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6061 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6121 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6181 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6241 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6301 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6361 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6421 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc gtttcctgca 6481 tcttttaatc aaatcccaag atgtgtataa acgcgccggt atgtacagga agaggtttat 6541 actaaactgt tacattgcaa acgtggtttc gtgtgccaag tgtgaaaacc gatgtttaat 6601 caaggctctg acgcatttct acaaccacga ctccaagtgt gtgggtgaag tcatgcatct 6661 tttaatcaaa tcccaagatg tgtataaacc accaaactgc caaaaaatga aaactgtcga 6721 caagctctgt ccgtttgctg gcaactgcaa gggtctcaat cctatttgta attattgaat 6781 aataaaacaa ttataaatgt caaatttgtt ttttattaac gatacaaacc aaacgcaaca 6841 agaacatttg tagtattatc tataattgaa aacgcgtagt tataatcgct gaggtaatat 6901 ttaaaatcat tttcaaatga ttcacagtta atttgcgaca atataatttt attttcacat 6961 aaactagacg ccttgtcgtc ttcttcttcg tattccttct ctttttcatt tttctcttca 7021 taaaaattaa catagttatt atcgtatcca tatatgtatc tatcgtatag agtaaatttt 7081 ttgttgtcat aaatatatat gtctttttta atggggtgta tagtaccgct gcgcatagtt 7141 tttctgtaat ttacaacagt gctattttct ggtagttctt cggagtgtgt tgctttaatt 7201 attaaattta tataatcaat gaatttggga tcgtcggttt tgtacaatat gttgccggca 7261 tagtacgcag cttcttctag ttcaattaca ccatttttta gcagcaccgg attaacataa 7321 ctttccaaaa tgttgtacga accgttaaac aaaaacagtt cacctccctt ttctatacta 7381 ttgtctgcga gcagttgttt gttgttaaaa ataacagcca ttgtaatgag acgcacaaac 7441 taatatcaca aactggaaat gtctatcaat atatagttgc tgatggccgg cctattaata 7501 gtaatcaatt acggggtc //