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. ACCESSION . VERSION . KEYWORDS CD8-mCherry-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 64..367 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 368..571 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 522..542 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 1385..1394 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1391..2095 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELY" primer_bind complement(1536..1554) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1804..1823 /label=mCherry-F /note="mCherry, forward primer" CDS 2099..2125 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2144..2422 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK" CDS 2156..2425 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ" polyA_signal 2548..2669 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2585..2604) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2639..2658 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2676..3131) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2813..2834) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3025..3044 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3158..3262 /gene="bla" /label=AmpR promoter promoter 3264..3621 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3287..3307) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3472..3607 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3534..3553 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3656..4450 /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(3710..3729) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4320..4339 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4638..4657) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4682..4729 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5058..5646 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5547..5566 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt 61 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc 121 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 181 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 241 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 301 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 361 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg 421 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca 481 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg 541 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg 601 ctaccggact cagatctcga gctcaagctt cgaattcggc ggctcccgcg cgcctcccct 661 cgcgcccgag cttcgagcca agcagcgtcc tggggagcgc gtcatggcct taccagtgac 721 cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccgagcc agttccgggt 781 gtcgccgctg gatcggacct ggaacctggg cgagacagtg gagctgaagt gccaggtgct 841 gctgtccaac ccgacgtcgg gctgctcgtg gctcttccag ccgcgcggcg ccgccgccag 901 tcccaccttc ctcctatacc tctcccaaaa caagcccaag gcggccgagg ggctggacac 961 ccagcggttc tcgggcaaga ggttggggga caccttcgtc ctcaccctga gcgacttccg 1021 ccgagagaac gagggctact atttctgctc ggccctgagc aactccatca tgtacttcag 1081 ccacttcgtg ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc cgcgaccacc 1141 aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc 1201 agcggcgggg ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg 1261 ggcgcccttg gccgggactt gtggagtcct tctcctgtca ctggttatca ccctttactg 1321 caagcggctt aagcgaagaa ggatccccgc cgccgccgcc gccgccgccg ccgtcagacc 1381 ggtcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 1441 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 1501 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 1561 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 1621 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 1681 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 1741 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 1801 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc 1861 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 1921 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1981 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga 2041 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacactta 2101 cccctacgac gtgcccgact acgcaggctc cgcacgagtg gccatcctct ggcatgagat 2161 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg 2221 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa 2281 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag 2341 gaagtacatg aaatcaggga atgtcaagga cctcacccaa gcctgggacc tctattatca 2401 tgtgttccga cgaatctcaa agcaggccgg ctgataatct agatcataat cagccatacc 2461 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 2521 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 2581 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 2641 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt 2701 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 2761 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 2821 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 2881 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 2941 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 3001 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 3061 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 3121 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3181 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 3241 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag 3301 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 3361 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3421 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3481 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3541 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3601 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat 3661 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 3721 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 3781 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga 3841 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 3901 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 3961 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 4021 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 4081 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 4141 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga 4201 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 4261 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 4321 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 4381 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 4441 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca 4501 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 4561 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 4621 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg 4681 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg 4741 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac 4801 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc 4861 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag 4921 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 4981 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 5041 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 5101 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 5161 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 5221 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 5281 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 5341 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 5401 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 5461 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 5521 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5581 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5641 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 5701 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatg //