LOCUS       sequence-193978-        6364 bp    DNA     circular SYN 15-DEC-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 6364)
  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 6364)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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-epsin(144-575)-GFP
FEATURES             Location/Qualifiers
     source          1..6364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     CDS             join(665..667,668..988)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      2303..2312
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2309..2311,2312..2314,2315..3028)
                     /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(2309..2311,2312..2314,2315..3025)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2354..2375)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2615..2634)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2962..2983
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3151..3272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3188..3207)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3242..3261
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3279..3734)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3416..3437)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3628..3647
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3761..3865
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3867..4224
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3867..4058
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3890..3910)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4075..4210
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4259..5053
                     /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             4259..5053
                     /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(4313..4332)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4923..4942
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5241..5260)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5285..5332
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5661..6249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6150..6169
                     /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 cggactcaga tctcgagaga tcgatgtgaa tgggggccct gaacccgcca agaacctgta
      661 caccatggga gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg
      721 cggccagacc tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aatttgattc
      781 ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg
      841 ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc
      901 agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt
      961 cttcgatgtg gagcttctaa aactggaacg gtaccatcgc gcccacgcgc tcaagaccaa
     1021 ggagaagctg gcacagactg ccacggcctc ctcagcagcc gtgggctctg gaccacctcc
     1081 cgaggcggag caggcatggc cacagagcag cggggaggag gagttgcagc tccagctggc
     1141 cctggctatg agcaaggagg aggccgacca gcccccgtcc tgcggccctg aggatgacgt
     1201 ccagctccag ctggccctta gtttgagccg agaggagcac gataaggaag agcggatccg
     1261 tcgtggggat gacctgaggc tgcagatggc aatagaggag agcaagaggg agactggggg
     1321 caaggaggag tcatctctca tggatcttgc tgatgtcttc acaaccccag cgcctccaca
     1381 ggcctcagac ccttgggggg gcccggcatc tgtgcctact gctgtccctg tggctgctgc
     1441 cgcttcagac ccttgggggg gacctgctgt ccctccagct gctgatcctt ggggtggtgc
     1501 agcccccaca ccagcctcag gggatccctg gaggcctgca gcccctacag ggccctctgt
     1561 tgatccttgg ggtgggaccc cagctcctgc agctggagag gggcccaccc ctgacccgtg
     1621 gggaagctct gatggtgggg ctcctgtcag tgggccccca tcttctgacc cctgggcacc
     1681 tgccccggct ttctcagacc cctggggagg ctcacctgca aagcccagca gcaacggcac
     1741 tgcagttggg ggctttgaca cagagcctga tgagttctca gacttcgacc gactccgcac
     1801 tgccctgcca acctctggga gcagcacggg ggagttggag ttgctcgcag gagaggtgcc
     1861 tgctcgcagc cctggggcat tcgacatgag tggggttggg ggatctctgg ctgagtctgt
     1921 ggggagtccc ccacctgctg caaccccaac tcccaccccc ccaacacgga agactccgga
     1981 gtcattccta ggccctaatg cagcccttgt ggatctggac tcgctggtga gccgaccagg
     2041 ccccacacca ccaggatcca aggcttccaa cccattcctt ccaagcggag ctccgcccac
     2101 aggcccctct gtcaccaatc ctttccagcc agcaccccct gcaacgctca ccctgaacca
     2161 gctccgtctc agtcctgtgc ccccagttcc tggagcgcca cccacctata tctctcctct
     2221 tggtggaggc cctggcctgc cccccatgat gcccccaggt cccccagccc ccaacactaa
     2281 ccccttcctc ctaagaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     2341 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2401 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2461 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2521 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2581 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2641 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2701 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2761 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2821 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2881 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2941 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3001 tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat
     3061 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     3121 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     3181 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3241 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     3301 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3361 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3421 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3481 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3541 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3601 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3661 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3721 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3781 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3841 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     3901 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3961 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4021 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4081 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4141 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4201 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     4261 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4321 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4381 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4441 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4501 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4561 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4621 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4681 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4741 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     4801 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4861 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4921 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4981 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5041 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5101 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5161 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5221 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     5281 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     5341 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     5401 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     5461 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     5521 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5581 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5641 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5701 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5761 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5821 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5881 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5941 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6001 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6061 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6121 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6181 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6241 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6301 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     6361 catt
//
