LOCUS       sequence-193982-        6222 bp    DNA     circular SYN 14-APR-2025
DEFINITION  To express a clathrin ""hook"" in mammalian cells to hot-wire 
            endocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6222)
  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 6222)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2017 Sep 27. pii: jcb.201702188. doi: 10.1083/jcb.201702188."
COMMENT     Sequence Label: FKBP-beta3(702-1094)-GFP
FEATURES             Location/Qualifiers
     source          1..6222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        47..350
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        351..554
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     505..525
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(621..623,624..944)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      2148..2157
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2154..2156,2157..2159,2160..2873)
                     /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             join(2154..2156,2157..2159,2160..2870)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2199..2220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2460..2479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2807..2828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2996..3117
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3033..3052)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3087..3106
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3124..3579)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3261..3282)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3473..3492
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3606..3710
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3712..4069
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3712..3903
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3735..3755)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3920..4055
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4104..4898
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4104..4898
                     /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(4158..4177)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4768..4787
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5086..5105)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5130..5177
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5506..6094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5995..6014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
       61 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      121 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      181 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      241 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      301 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      361 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      421 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      481 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      541 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg actcagatct
      601 cgagctcaag cttcgaattc atgggagtgc aggtggaaac catctcccca ggagacgggc
      661 gcaccttccc caagcgcggc cagacctgcg tggtgcacta caccgggatg cttgaagatg
      721 gaaagaaatt tgattcctcc cgggacagaa acaagccctt taagtttatg ctaggcaagc
      781 aggaggtgat ccgaggctgg gaagaagggg ttgcccagat gagtgtgggt cagagagcca
      841 aactgactat atctccagat tatgcctatg gtgccactgg gcacccaggc atcatcccac
      901 cacatgccac tctcgtcttc gatgtggagc ttctaaaact ggaacggtac cgcgggcccg
      961 aacaaggcga aagtggggag gaaggagaca gcaatgagga cagcagtgag gactcctcca
     1021 gtgagcagga cagtgagagt ggacgggagt caggcctaga aaacaaaaga acagccaaga
     1081 ggaactcaaa agccaaagga aaaagtgatt ctgaagatgg ggagaaggaa aatgaaaaat
     1141 ctaaaacttc agattcttca aatgacgaat ctagttcaat agaagacagt tcttccgatt
     1201 ctgaatcaga gtcagaacct gaaagtgaat ctgaatccag aagagtcact aaggagaaag
     1261 aaaagaaaac aaagcaagat agaactcctc ttaccaaaga tgtttcactt ctagatctgg
     1321 atgattttaa cccagtatcc actccagttg cacttcccac accagctctt tctccaagtt
     1381 tgatggctga tcttgaaggt ttacacttgt caacttcctc ttcagtcatc agtgtcagta
     1441 ctcctgcatt tgtaccaacg aaaactcacg tgctgcttca tcgaatgagt ggaaaaggac
     1501 tagctgccca ttatttcttt ccaagacagc cttgcatttt tggtgataag atggtctcta
     1561 tacaaataac actgaataac actactgatc gaaagataga aaatatccac ataggggaaa
     1621 aaaaacttcc tataggcatg aaaatgcatg tttttaatcc aatagactct cttgagcctg
     1681 agggatccat tacagtttca atgggtattg acttttgtga ttctactcag actgccagtt
     1741 tccagttgtg taccaaggat gattgcttca atgttaatat tcagccacct gttggagaac
     1801 tgcttttacc tgtggccatg tcagagaaag attttaagaa agagcaagga gtgctaacag
     1861 gaatgaatga aacttctgct gtaatcattg ctgcaccaca gaatttcact ccctctgtga
     1921 tctttcagaa ggttgtaaat gtagccaatg taggtgcagt cccttctggc caggataata
     1981 tacacaggtt tgcagctaaa actgtgcaca gtgggtcatt gatgctagtc acagtggaac
     2041 tgaaggaagg ctctacagcc cagcttatca taaacactga gaaaactgtg attggctctg
     2101 ttctgctgcg ggaactgaag cctgtcctgt ctcaggggtc accggtcgcc accatggtga
     2161 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2221 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2281 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2341 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2401 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2461 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2521 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2581 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2641 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2701 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2761 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2821 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc
     2881 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     2941 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3001 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3061 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     3121 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3181 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3241 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3301 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3361 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3421 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3481 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3541 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     3601 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3661 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     3721 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3781 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3841 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3901 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3961 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4021 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     4081 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     4141 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4201 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4261 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     4321 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4381 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4441 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4501 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4561 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4621 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4681 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4741 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4801 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4861 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4921 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4981 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5041 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     5101 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     5161 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5221 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5281 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5341 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5401 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5461 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5521 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5581 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5641 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6121 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6181 tgtggataac cgtattaccg ccatgcatta gttattaata gt
//
