LOCUS Exported 5259 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PI(3,4)P2 biosensor. ACCESSION . VERSION . KEYWORDS NES-EGFP-cPHx1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5259) AUTHORS Goulden BD, Pacheco J, Dull A, Zewe JP, Deiters A, Hammond GRV TITLE A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is predominantly a class I PI3K signaling product. JOURNAL J Cell Biol. 2019 Mar 4;218(3):1066-1079. doi: 10.1083/jcb.201809026. Epub 2018 Dec 27. PUBMED 30591513 REFERENCE 2 (bases 1 to 5259) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5259 /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 535..544 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 592..601 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 598..1314 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 598..1314 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(643..664) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(904..923) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1251..1272 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 1987..2108 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2024..2043) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2078..2097 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2115..2570) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2252..2273) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2464..2483 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2597..2701 /gene="bla" /label=AmpR promoter promoter 2703..3060 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2726..2746) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2911..3046 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2973..2992 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3095..3889 /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(3149..3168) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3759..3778 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4077..4096) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4121..4168 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 4497..5085 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4986..5005 /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 gctagccacc 541 atggctctgc agaaaaagtt ggaagagctt gagctggatg aggcaccggt cgccaccatg 601 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 661 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 721 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 781 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 841 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 901 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 961 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 1021 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 1081 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 1141 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 1201 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1261 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga 1321 ctcagatctc gagctcaagc ttcgaattct agaaataatc tgaaacggtc acaaagccat 1381 cttccttact ttactcctaa accacctcaa gatagtgcgg ttatcaaagc tggatattgt 1441 gtaaaacaag gagcagtgat gaaaaactgg aagagaagat attttcaatt ggatgaaaac 1501 acaataggct acttcaaatc tgaactggaa aaggaacctc ttcgtgtaat accacttaaa 1561 gaggttcata aagtccagga atgtaagcaa agcgacataa tgatgaggga caacctcttt 1621 gaaattgtaa caacgtctcg aactttctat gtgcaggctg atagccctga agagatgcac 1681 agttggatta aagcagtctc tggcgccatt gtagcacagc ggggtcccgg cagatctgcg 1741 tcttctgagc atccccccgg tccttcagaa tccaaacacg ctttccgtcc taccaacgca 1801 gccaccgcca cctcacattc cacagcctct cgctgagtcg acggtaccgc gggcccggga 1861 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt 1921 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 1981 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 2041 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 2101 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg 2161 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 2221 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 2281 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 2341 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 2401 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 2461 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 2521 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac 2581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 2641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 2701 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 2761 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 2821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 2881 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 2941 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 3001 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 3061 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc 3121 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 3181 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 3241 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg 3301 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 3361 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 3421 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 3481 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 3541 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 3601 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg 3661 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 3721 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 3781 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 3841 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg 3901 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc 3961 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc 4021 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa 4081 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat 4141 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac 4201 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc 4261 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca 4321 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt 4381 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4621 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 4681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 5041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 5101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 5161 tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat 5221 tacggggtca ttagttcata gcccatatat ggagttccg //