LOCUS       sequence-189375-        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        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             join(666..668,669..989)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      2304..2313
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2310..2312,2313..2315,2316..3029)
                     /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(2310..2312,2313..2315,2316..3026)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2355..2376)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2616..2635)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2963..2984
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3152..3273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3189..3208)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3243..3262
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3280..3735)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3417..3438)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3629..3648
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3762..3866
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3868..4225
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3868..4059
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3891..3911)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4076..4211
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4260..5054
                     /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             4260..5054
                     /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(4314..4333)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4924..4943
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5242..5261)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5286..5333
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5662..6250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6151..6170
                     /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 ggtaccatcg cgcccacgcg ctcaagacca
     1021 aggagaagct ggcacagact gccacggcct cctcagcagc cgtgggctct ggaccacctc
     1081 ccgaggcgga gcaggcatgg ccacagagca gcggggagga ggagttgcag ctccagctgg
     1141 ccctggctat gagcaaggag gaggccgacc agcccccgtc ctgcggccct gaggatgacg
     1201 tccagctcca gctggccctt agtttgagcc gagaggagca cgataaggaa gagcggatcc
     1261 gtcgtgggga tgacctgagg ctgcagatgg caatagagga gagcaagagg gagactgggg
     1321 gcaaggagga gtcatctctc atggatcttg ctgatgtctt cacaacccca gcgcctccac
     1381 aggcctcaga cccttggggg ggcccggcat ctgtgcctac tgctgtccct gtggctgctg
     1441 ccgcttcaga cccttggggg ggacctgctg tccctccagc tgctgatcct tggggtggtg
     1501 cagcccccac accagcctca ggggatccct ggaggcctgc agcccctaca gggccctctg
     1561 ttgatccttg gggtgggacc ccagctcctg cagctggaga ggggcccacc cctgacccgt
     1621 ggggaagctc tgatggtggg gctcctgtca gtgggccccc atcttctgac ccctgggcac
     1681 ctgccccggc tttctcagac ccctggggag gctcacctgc aaagcccagc agcaacggca
     1741 ctgcagttgg gggctttgac acagagcctg atgagttctc agacttcgac cgactccgca
     1801 ctgccctgcc aacctctggg agcagcacgg gggagttgga gttgctcgca ggagaggtgc
     1861 ctgctcgcag ccctggggca ttcgacatga gtggggttgg gggatctctg gctgagtctg
     1921 tggggagtcc cccacctgct gcaaccccaa ctcccacccc cccaacacgg aagactccgg
     1981 agtcattcct aggccctaat gcagcccttg tggatctgga ctcgctggtg agccgaccag
     2041 gccccacacc accaggatcc aaggcttcca acccattcct tccaagcgga gctccgccca
     2101 caggcccctc tgtcaccaat cctttccagc cagcaccccc tgcaacgctc accctgaacc
     2161 agctccgtct cagtcctgtg cccccagttc ctggagcgcc acccacctat atctctcctc
     2221 ttggtggagg ccctggcctg ccccccatga tgcccccagg tcccccagcc cccaacacta
     2281 accccttcct cctaagaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     2341 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2401 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2461 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2521 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2581 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2641 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2701 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2761 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2821 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2881 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2941 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     3001 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     3061 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3121 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3181 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3241 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     3301 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3361 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3421 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3481 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3541 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3601 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3661 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3721 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3781 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3841 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     3901 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3961 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4021 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4081 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4141 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4201 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     4261 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4321 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4381 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4441 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4501 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4561 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4621 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4681 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4741 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4801 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4861 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4921 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4981 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5041 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5101 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5161 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5221 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     5281 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     5341 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     5401 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     5461 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     5521 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5581 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5641 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5701 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5761 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5821 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5881 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5941 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6001 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6061 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6301 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6361 gcat
//
