LOCUS Exported 7096 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Yellow/red FRET biosensor for monotoring plasmamembrane PtdIns(4,5)P2 levels (PIP2). ACCESSION . VERSION . KEYWORDS PH-PLCD1_mScarlet_IRES_SYFP2_PH_N1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7096) 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 7096) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7096 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 1102..1111 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1108..1803 /codon_start=1 /product="bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016)" /label=mScarlet /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGMDELYK" primer_bind complement(1241..1259) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1509..1528 /label=mCherry-F /note="mCherry, forward primer" promoter complement(1831..1848) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1831..1848) /label=SP6 /note="SP6 promoter, forward primer" misc_feature 1860..2433 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 1883..2435 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2027..2044) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2254..2273 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 2462..2471 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2468..3184 /codon_start=1 /product="monomeric optimized variant of EYFP, identical to mVenus but with L68V (Kremers et al., 2006)" /label=SYFP2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2513..2534) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2774..2793) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3121..3142 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 3824..3945 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3861..3880) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3915..3934 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(3952..4407) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4089..4110) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4301..4320 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4434..4538 /gene="bla" /label=AmpR promoter promoter 4540..4897 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(4563..4583) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4748..4883 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4810..4829 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4932..5726 /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(4986..5005) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5596..5615 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5914..5933) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5958..6005 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 6334..6922 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6823..6842 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 541 ccggactcag atctcgagct caagcttcga attcacggca tggactcggg ccgggacttc 601 ctgaccctgc acggcctaca ggatgatgag gatctacagg cgctgctgaa gggcagccag 661 ctcctgaagg tgaagtccag ctcatggagg agagagcgct tctacaagtt gcaggaggac 721 tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc ccagctgttc 781 tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct ggagaagttc 841 gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca gcgcaataca 901 ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg gctgcacaag 961 atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg gattcactcc 1021 tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga gctgcagaac 1081 ttcctgaagg atccaccggt cgccaccatg gtgagcaagg gcgaggcagt gatcaaggag 1141 ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag 1201 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag 1261 ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg 1321 gccttcacca agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc 1381 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac 1441 acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct 1501 cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg 1561 taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc 1621 ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc 1681 ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg 1741 gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac 1801 aagtccggat aacgcgacag atctgggccc ctatagtgtc acctaaataa ttccgccccc 1861 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt 1921 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg 1981 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga 2041 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa 2101 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc 2161 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac 2221 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag 2281 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc 2341 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg 2401 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacagcgg atccaccggt 2461 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 2521 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 2581 cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg 2641 gcccaccctc gtgaccaccc tgggctacgg cgtgcagtgc ttcgcccgct accccgacca 2701 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 2761 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 2821 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 2881 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca 2941 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca 3001 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 3061 caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca 3121 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 3181 caagtccgga ctcagatctc gaggcctaca ggatgatgag gatctacagg cgctgctgaa 3241 gggcagccag ctcctgaagg tgaagtccag ctcatggagg agagagcgct tctacaagtt 3301 gcaggaggac tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc 3361 ccagctgttc tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct 3421 ggagaagttc gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca 3481 gcgcaataca ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg 3541 gctgcacaag atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg 3601 gattcactcc tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga 3661 gctgcagaac ttcctgaagg agctcaacat ctgaggatcc accggatcta gataactgat 3721 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 3781 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 3841 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3901 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa 3961 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 4021 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 4081 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 4141 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 4201 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 4261 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 4321 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg 4381 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa 4441 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4501 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct 4561 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4621 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 4681 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 4741 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 4801 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 4861 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag 4921 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 4981 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 5041 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 5101 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 5161 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 5221 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 5281 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 5341 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 5401 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga 5461 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 5521 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 5581 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 5641 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 5701 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc 5761 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg 5821 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 5881 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga 5941 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt 6001 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc 6061 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg 6121 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta 6181 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 6241 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 6301 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 6361 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttttttgc cggatcaaga 6421 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 6481 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 6541 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 6601 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 6661 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 6721 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6781 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6841 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6901 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 6961 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 7021 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc 7081 catatatgga gttccg //