LOCUS Exported 7096 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION It expresses separate mScarletI pHdomain and SYFP2 pHdomain in a 3:1 ratio.. ACCESSION . VERSION . KEYWORDS PH-PLCD1_mScarlet-I_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 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 1162..1171 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1168..1863 /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(1301..1319) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1569..1588 /label=mCherry-F /note="mCherry, forward primer" promoter complement(1891..1908) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1891..1908) /label=SP6 /note="SP6 promoter, forward primer" misc_feature 1920..2493 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 1943..2495 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2087..2104) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2314..2333 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 2522..2531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2528..3244 /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(2573..2594) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2834..2853) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3181..3202 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 3884..4005 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3921..3940) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3975..3994 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(4012..4467) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4149..4170) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4361..4380 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4494..4598 /gene="bla" /label=AmpR promoter promoter 4600..4957 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(4623..4643) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4808..4943 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4870..4889 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4992..5786 /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(5046..5065) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5656..5675 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5974..5993) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6018..6065 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 6394..6982 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6883..6902 /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 ccggactcag atctcgagct caagcttcga attcacggca tggactcggg ccgggacttc 661 ctgaccctgc acggcctaca ggatgatgag gatctacagg cgctgctgaa gggcagccag 721 ctcctgaagg tgaagtccag ctcatggagg agagagcgct tctacaagtt gcaggaggac 781 tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc ccagctgttc 841 tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct ggagaagttc 901 gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca gcgcaataca 961 ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg gctgcacaag 1021 atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg gattcactcc 1081 tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga gctgcagaac 1141 ttcctgaagg atccaccggt cgccaccatg gtgagcaagg gcgaggcagt gatcaaggag 1201 ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag 1261 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag 1321 ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg 1381 gccttcatca agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc 1441 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac 1501 acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct 1561 cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg 1621 taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc 1681 ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc 1741 ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg 1801 gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac 1861 aagtccggat aacgcgacag atctgggccc ctatagtgtc acctaaataa ttccgccccc 1921 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt 1981 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg 2041 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga 2101 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa 2161 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc 2221 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac 2281 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag 2341 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc 2401 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg 2461 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacagcgg atccaccggt 2521 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 2581 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 2641 cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg 2701 gcccaccctc gtgaccaccc tgggctacgg cgtgcagtgc ttcgcccgct accccgacca 2761 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 2821 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 2881 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 2941 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca 3001 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca 3061 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 3121 caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca 3181 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 3241 caagtccgga ctcagatctc gaggcctaca ggatgatgag gatctacagg cgctgctgaa 3301 gggcagccag ctcctgaagg tgaagtccag ctcatggagg agagagcgct tctacaagtt 3361 gcaggaggac tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc 3421 ccagctgttc tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct 3481 ggagaagttc gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca 3541 gcgcaataca ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg 3601 gctgcacaag atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg 3661 gattcactcc tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga 3721 gctgcagaac ttcctgaagg agctcaacat ctgaggatcc accggatcta gataactgat 3781 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 3841 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 3901 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3961 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa 4021 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 4081 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 4141 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 4201 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 4261 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 4321 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 4381 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg 4441 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa 4501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4561 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct 4621 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4681 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 4741 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 4801 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 4861 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 4921 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag 4981 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 5041 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 5101 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 5161 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 5221 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 5281 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 5341 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 5401 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 5461 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga 5521 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 5581 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 5641 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 5701 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 5761 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc 5821 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg 5881 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 5941 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga 6001 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt 6061 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc 6121 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg 6181 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta 6241 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 6301 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 6361 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 6421 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 6481 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 6541 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 6601 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 6661 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 6721 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 6781 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6841 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6901 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6961 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 7021 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 7081 tattaccgcc atgcat //