LOCUS Exported 7554 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION p115RhoGEF tagged with mVenus for detection of Ga13 activity. ACCESSION . VERSION . KEYWORDS mVenus-P115-C1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7554) AUTHORS Mastop M, Reinhard NR, Zuconelli CR, Terwey F, Gadella TWJ Jr, van Unen J, Adjobo-Hermans MJW, Goedhart J TITLE A FRET-based biosensor for measuring Galpha13 activation in single cells. JOURNAL PLoS One. 2018 Mar 5;13(3):e0193705. doi: 10.1371/journal.pone.0193705. eCollection 2018. PUBMED 29505611 REFERENCE 2 (bases 1 to 7554) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7554 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 61..364 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 365..568 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 519..539 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 607..616 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 613..1329 /codon_start=1 /product="Venus YFP with monomerizing A206K mutation (Nagai et al., 2002; Kremers et al., 2006)" /label=mVenus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(658..679) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(919..938) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1266..1287 /label=EGFP-C /note="EGFP, forward primer" protein_bind 1408..1432 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" polyA_signal 4342..4463 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4379..4398) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4433..4452 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(4470..4925) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4607..4628) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4819..4838 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4952..5056 /gene="bla" /label=AmpR promoter promoter 5058..5415 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(5081..5101) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 5266..5401 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5328..5347 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5450..6244 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5504..5523) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6114..6133 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(6432..6451) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6476..6523 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 6852..7440 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7341..7360 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 721 gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg 781 ccctggccca ccctcgtgac caccctgggc tacggcctgc agtgcttcgc ccgctacccc 841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac 1081 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc 1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1201 cccgacaacc actacctgag ctaccagtcc aagctgagca aagaccccaa cgagaagcgc 1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1321 ctgtacaagt ccggactcag atctcttggt accgagctcg gatccactag taacggccgc 1381 cagtgtgctg gaattctgca gatatcaaca agtttgtaca aaaaagcagg ctccgcggcc 1441 gcccccttca ccatggaaga cttcgcccga ggggcggcct ccccaggccc ctcccggcct 1501 ggcctggttc ccgtcagcat catcggggct gaggatgagg attttgagaa cgagctggag 1561 acaaactcag aagagcaaaa cagccagttc cagagcctgg agcaggtgaa gcggcgccca 1621 gcccacctca tggccctcct gcagcacgtg gccctgcagt ttgagccagg acccctgctt 1681 tgctgtctgc atgccgacat gctgggctca ctgggcccca aggaggccaa gaaggccttc 1741 ctggacttct accacagctt cctggagaag acagcggttc tccgggtgcc ggtccctccc 1801 aacgtcgcct ttgaacttga ccgcactagg gctgacctca tctccgagga tgtccagcgg 1861 cggttcgtgc aggaggtggt gcaaagccag caggtagccg tgggccggca gctggaggac 1921 ttccgttcca agcggctcat gggcatgacg ccctgggagc aggagctggc ccagctggag 1981 gcttgggttg ggcgggaccg agccagctac gaggcccggg agcggcacgt ggcggagcgg 2041 ctgctcatgc acctggagga gatgcaacat accatctcta ccgacgaaga aaagagtgct 2101 gccgtggtca acgccattgg cctgtacatg cgccaccttg gggtgcggac caagagtgga 2161 gacaagaagt cggggaggaa cttcttccgg aaaaaggtga tggggaaccg gcggtcggac 2221 gagcctgcca agaccaagaa ggggctgagc agcatcctgg atgccgcccg ctggaaccgg 2281 ggagagcccc aggttccaga ttttcgacac ctcaaagcag aggttgatgc cgagaagcca 2341 ggtgctacag accggaaggg aggcgtgggg atgccctctc gggaccggaa tatcggggct 2401 cctgggcagg acacccctgg agtctctctg caccctctgt ccctggacag cccagaccgg 2461 gaaccaggtg ctgacgcccc cctggagctg ggggactcat ccccgcaggg cccaatgagc 2521 ctggagtcct tggcgccccc agagagtacc gacgaggggg ccgaaaccga gagccccgag 2581 cctggagatg agggggagcc ggggcggtcg ggactggagc ttgaaccaga agagcctccc 2641 ggctggcggg aactcgtccc cccagacacc ctgcacagcc tgcccaagag ccaggtgaag 2701 cggcaggagg tcatcagcga gctgctggtg acagaggcgg cccacgtgcg catgctgcgg 2761 gtgctgcacg acctcttctt ccagcccatg gcagaatgcc tgttcttccc cttggaggag 2821 ctgcagaaca tcttccccag cctggacgag ctcatcgagg tgcattccct gttcctcgat 2881 cgcctgatga agcggaggca ggagagtggc tacctcatcg aggagatcgg agacgtgctg 2941 ctggcccggt ttgatggtgc tgagggctcc tggttccaga aaatctcctc ccgcttctgc 3001 agccgccagt catttgcctt agagcagctc aaagccaagc aacgcaagga ccctcggttc 3061 tgtgccttcg tgcaggaagc tgagagccgc ccgcggtgcc gccgcctgca gctgaaggac 3121 atgatcccca cggagatgca gcggctgacc aagtaccccc tgctcctgca gagcatcggg 3181 cagaacacag aagagcccac agaacgggag aaagtggagc tggcagccga gtgctgccgg 3241 gaaattctac accacgtcaa ccaagccgtg cgtgacatgg aggacctgct gaggctcaag 3301 gactatcagc ggcgcctgga cttgtcccac cttcggcaga gcagcgaccc tatgctgagc 3361 gagttcaaga acctggacat caccaagaag aaattggtcc acgagggccc actgacgtgg 3421 cgggtgacta aggacaaggc agtggaggtg catgtgctgc tgctggacga cctgctgctg 3481 ctgctccagc gccaggacga gcggctgctg ctcaagtccc atagccggac actgacgccc 3541 acgcccgatg gcaagaccat gctgcggccc gtgctgcggc tcacctccgc catgacccgc 3601 gaggtggcca ccgatcacaa agccttctac gtccttttta cctgggacca ggaggcccag 3661 atatacgagc tggtggcaca gactgtgtcg gagcggaaaa actggtgtgc tctcatcact 3721 gagactgccg gatccctgaa agtccctgcc cctgcctctc gccctaagcc ccggcccagc 3781 ccgagcagca cccgagaacc cctcctcagc agctctgaga acggcaatgg tggccgagag 3841 acgtctccag ctgatgcccg gaccgagaga atcctcagtg acctcctgcc cttctgcaga 3901 ccaggccccg agggccagct cgctgccacg gcccttcgga aagtgctgtc cctgaagcag 3961 cttctgtttc cggcggagga agacaatggg gcggggcctc ctcgagatgg ggatggggtc 4021 ccagggggcg gccccctgag cccagcacgg acccaggaaa tccaggagaa cctgctcagc 4081 ttggaggaga ccatgaagca gctggaggag ttggaggagg aattttgccg cctgagaccc 4141 ctcctgtctc agcttggggg gaactctgtc ccccagcctg gctgcacttg agtcgacggt 4201 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt 4261 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 4321 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 4381 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 4441 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt 4501 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 4561 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 4621 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 4681 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 4741 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 4801 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 4861 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 4921 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 4981 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 5041 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg 5101 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 5161 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 5221 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 5281 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 5341 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 5401 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca 5461 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 5521 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 5581 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc 5641 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 5701 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 5761 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 5821 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 5881 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 5941 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct 6001 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 6061 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 6121 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 6181 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 6241 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 6301 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 6361 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg 6421 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca 6481 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg 6541 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc 6601 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc 6661 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat 6721 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 6781 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 6841 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 6901 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 6961 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 7021 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 7081 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 7141 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 7201 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 7261 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 7321 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 7381 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 7441 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 7501 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat //