LOCUS Exported 6073 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION To express a clathrin ""hook"" mutant in mammalian cells to hot-wire endocytosis. ACCESSION . VERSION . KEYWORDS FKBP-beta2(616-951)-GFP Double SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6073) 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 6073) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6073 /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" CDS 666..989 /codon_start=1 /product="human FK506-binding protein FKBP12" /label=FKBP /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL LKLE" regulatory 1014..1023 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 2013..2022 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2019..2738 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2019..2735 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2064..2085) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2325..2344) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2672..2693 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2861..2982 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2898..2917) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2952..2971 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2989..3444) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3126..3147) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3338..3357 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3471..3575 /gene="bla" /label=AmpR promoter promoter 3577..3934 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3600..3620) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3785..3920 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3847..3866 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3969..4763 /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(4023..4042) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4633..4652 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4951..4970) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4995..5042 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5371..5959 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5860..5879 /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 atctcgagag atcgatgtga atgggggccc tgaacccgcc aagaacctgt 661 acaccatggg agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc ttccccaagc 721 gcggccagac ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaatttgatt 781 cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag 841 gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc 901 cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg 961 tcttcgatgt ggagcttcta aaactggaac ggtaccgcgg gcccgggatc cccgccacca 1021 tggaacagcc tcaggttatc ccctctcaag gtgatctcgg tcccccagtc aatgtgccac 1081 aggtgtcctc catgcagatg ggagcagtgg atctcctagg aggaggacta gatagtctgc 1141 ttggcagtga ccttggcggg ggcattggag gaagtccggc agtgggacaa tccttcatcc 1201 catcatcggt ccctgcaacc tttgctcctt cacctacacc tgctgtggtc agcagtggac 1261 tgaatgacct gtttgaactc tccacaggga taggcatggc acctggtgga tatgtggctc 1321 ctaaggctgt ctggctacct gcagtaaagg ctaaaggctt ggagatttcc ggaacattta 1381 ctcaccgcca agggcacatc tatatggaaa tgaacttcac caataaagct ctgcagcaca 1441 tgacagattt tgcaatccag tttaacaaaa atagctttgg tgtcatcccc agcactcctc 1501 tggccatcca tacaccactg atgccaaacc agagcattga tgtctccctg cctctcaata 1561 ccttgggccc agtcatgaag atggaacctc tgaataacct ccaggtggct gtgaaaaaca 1621 atatcgatgt cttcgccttc agctgcctca tcccactcaa tgtgcttttt gtagaagatg 1681 gcaaaatgga gcgccaggtc ttccttgcaa catggaagga tattcccaat gaaaatgaac 1741 ttcagtttca gattaaggaa tgtcatttaa atgctgacac tgtttccagc aagttgcaaa 1801 acaacaatgt ttatactatt gccaagagga atgtggaagg gcaggacatg ctgtaccaat 1861 ccctgaagct cactaatggc atttggattt tggccgaact acgtatccag ccaggaaacc 1921 ccaattacac gctgtcactg aagtgtagag ctcctgaagt ctctcaatac atctatcagg 1981 tctacgacag cattttgaaa aacttaccgg tcgccaccat ggtgagcaag ggcgaggagc 2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 2101 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 2161 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 2221 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 2281 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 2461 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 2521 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 2581 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 2641 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 2701 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat 2761 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 2821 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 2881 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 2941 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg 3001 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 3061 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 3121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 3181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 3241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag 3301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 3361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 3421 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 3481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 3541 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg 3601 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3661 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 3721 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 3781 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 3841 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 3901 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat 3961 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga 4021 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc 4081 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga 4141 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg 4201 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc 4261 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg 4321 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga 4381 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc 4441 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca 4501 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg 4561 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct 4621 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg 4681 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc 4741 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac 4801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 4861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 4921 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg 4981 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt 5041 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca 5101 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga 5161 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc 5221 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 5281 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5341 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5401 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5461 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 5521 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5581 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5641 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5701 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5761 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5821 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5881 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5941 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 6001 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 6061 taccgccatg cat //