LOCUS Exported 5208 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses GFP-tagged HRS 2xFYVE in mammalian cells. ACCESSION . VERSION . KEYWORDS pEGFP-2xFYVE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5208) AUTHORS Gillooly DJ, Morrow IC, Lindsay M, Gould R, Bryant NJ, Gaullier JM, Parton RG, Stenmark H TITLE Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. JOURNAL EMBO J. 2000 Sep 1;19(17):4577-88. doi: 10.1093/emboj/19.17.4577. PUBMED 10970851 REFERENCE 2 (bases 1 to 5208) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5208 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 60..363 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 364..567 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 518..538 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 606..615 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 612..1328 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 612..1328 /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(657..678) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(918..937) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1265..1286 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 1995..2116 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2032..2051) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2086..2105 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2123..2578) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2260..2281) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2472..2491 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2605..2709 /gene="bla" /label=AmpR promoter promoter 2711..3068 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2734..2754) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2919..3054 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2981..3000 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3103..3897 /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(3157..3176) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3767..3786 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4085..4104) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4129..4176 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 4505..5093 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4994..5013 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 361 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 541 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcgctac 601 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 661 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 721 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 781 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 841 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 901 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 961 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 1021 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 1081 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 1141 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 1201 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 1261 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 1321 tgtacaagta ctcagatctc gagctcaagc ttcgaattga attcgaaagt gatgccatgt 1381 tcgctgctga aagagcccct gactgggtgg atgctgagga atgccatcgg tgcagagtac 1441 agtttggggt ggtgacccgc aagcatcact gccgagcatg tgggcagatc ttctgtggca 1501 agtgctcctc caagtactcc accatcccca agttcggcat tgagaaggag gtgcgcgtgt 1561 gtgagccctg ctatgagcag ctgaacaaga aggcacaagg acaaggatcc gaaagtgatg 1621 ccatgttcgc tgctgaaaga gcccctgact gggtggatgc tgaggaatgc catcggtgca 1681 gagtacagtt tggggtggtg acccgcaagc atcactgccg agcatgtggg cagatcttct 1741 gtggcaagtg ctcctccaag tactccacca tccccaagtt cggcattgag aaggaggtgc 1801 gcgtgtgtga gccctgctat gagcagctga acaagaaggc ataagtcgac ggtaccgcgg 1861 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg 1921 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 1981 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2041 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 2101 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta 2161 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 2221 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 2281 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 2341 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 2401 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 2461 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 2521 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca 2581 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 2641 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 2701 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 2761 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2821 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 2881 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 2941 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 3001 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 3061 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 3121 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 3181 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 3241 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg 3301 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3361 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3421 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 3481 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 3541 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 3601 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg 3661 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 3721 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 3781 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 3841 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 3901 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 3961 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 4021 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc 4081 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa 4141 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg 4201 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt 4261 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg 4321 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac 4381 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 4441 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 4501 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4561 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4621 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 4681 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4741 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4801 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4861 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4921 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4981 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 5041 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 5101 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 5161 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcatt //