LOCUS Exported 5757 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To express a plasma membrane ""anchor"" in mammalian cells to hot-wire endocytosis (dark mCherry K70N). ACCESSION . VERSION . KEYWORDS CD8-dCherry-FRB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5757) AUTHORS Wood LA, Larocque G, Clarke NI, Sarkar S, Royle SJ TITLE New tools for ""hot-wiring"" clathrin-mediated endocytosis with temporal and spatial precision. JOURNAL J Cell Biol. 2017 Sep 27. pii: jcb.201702188. doi: 10.1083/jcb.201702188. PUBMED 28954824 REFERENCE 2 (bases 1 to 5757) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5757 /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 1382..1391 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1388..2092 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSNAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELY" primer_bind complement(1533..1551) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1801..1820 /label=mCherry-F /note="mCherry, forward primer" CDS 2096..2122 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2141..2419 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK" CDS 2153..2422 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ" polyA_signal 2545..2666 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2582..2601) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2636..2655 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2673..3128) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2810..2831) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3022..3041 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3155..3259 /gene="bla" /label=AmpR promoter promoter 3261..3618 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3284..3304) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3469..3604 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3531..3550 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3653..4447 /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(3707..3726) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4317..4336 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4635..4654) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4679..4726 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5055..5643 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5544..5563 /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 attcggcggc tcccgcgcgc ctcccctcgc 661 gcccgagctt cgagccaagc agcgtcctgg ggagcgcgtc atggccttac cagtgaccgc 721 cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccgagccagt tccgggtgtc 781 gccgctggat cggacctgga acctgggcga gacagtggag ctgaagtgcc aggtgctgct 841 gtccaacccg acgtcgggct gctcgtggct cttccagccg cgcggcgccg ccgccagtcc 901 caccttcctc ctatacctct cccaaaacaa gcccaaggcg gccgaggggc tggacaccca 961 gcggttctcg ggcaagaggt tgggggacac cttcgtcctc accctgagcg acttccgccg 1021 agagaacgag ggctactatt tctgctcggc cctgagcaac tccatcatgt acttcagcca 1081 cttcgtgccg gtcttcctgc cagcgaagcc caccacgacg ccagcgccgc gaccaccaac 1141 accggcgccc accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc 1201 ggcggggggc gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc 1261 gcccttggcc gggacttgtg gagtccttct cctgtcactg gttatcaccc tttactgcaa 1321 gcggcttaag cgaagaagga tccccgccgc cgccgccgcc gccgccgccg tcagaccggt 1381 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg 1441 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg 1501 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc 1561 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca acgcctacgt 1621 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg 1681 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct 1741 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg 1801 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga 1861 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta 1921 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta 1981 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca 2041 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acacttaccc 2101 ctacgacgtg cccgactacg caggctccgc acgagtggcc atcctctggc atgagatgtg 2161 gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg tgaaaggcat 2221 gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga ctctgaagga 2281 aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt ggtgcaggaa 2341 gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct attatcatgt 2401 gttccgacga atctcaaagc aggccggctg ataatctaga tcataatcag ccataccaca 2461 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat 2521 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa 2581 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 2641 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa 2701 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 2761 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 2821 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 2881 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 2941 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 3001 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 3061 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 3121 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 3181 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 3241 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta 3301 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 3361 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 3421 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 3481 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 3541 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 3601 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga 3661 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 3721 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 3781 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga 3841 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 3901 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 3961 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 4021 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 4081 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 4141 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga 4201 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 4261 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 4321 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 4381 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 4441 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca 4501 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 4561 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct 4621 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg 4681 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt 4741 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc 4801 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca 4861 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt 4921 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 4981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5161 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 5581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 5641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 5701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat //