LOCUS       sequence-193571-        6100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a clathrin ""hook"" mutant in mammalian cells to hot-wire
            endocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6100)
  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 6100)
  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-beta2(616-951)-GFP Y215A
FEATURES             Location/Qualifiers
     source          1..6100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..368
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        369..572
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     523..543
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(670..672,673..993)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      1018..1027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2044..2053
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2050..2052,2053..2055,2056..2769)
                     /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(2050..2052,2053..2055,2056..2766)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2095..2116)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2356..2375)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2703..2724
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2892..3013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2929..2948)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2983..3002
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3020..3475)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3157..3178)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3369..3388
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3502..3606
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3608..3965
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3608..3799
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3631..3651)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3816..3951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4000..4794
                     /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             4000..4794
                     /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(4054..4073)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4664..4683
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4982..5001)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5026..5073
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5402..5990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5891..5910
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
       61 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      121 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      181 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      241 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      301 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      361 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      421 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      481 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      541 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc
      601 gctaccggac tcagatctcg agagatcgat gtgaatgggg gccctgaacc cgccaagaac
      661 ctgtacacca tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc
      721 aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaattt
      781 gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc
      841 cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata
      901 tctccagatt atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact
      961 ctcgtcttcg atgtggagct tctaaaactg gaacggtacc gcgggcccgg gatccccgcc
     1021 accatggaac agcctcaggt tatcccctct caaggtgatc ttctagggga tcttttaaac
     1081 cttgacctcg gtcccccagt caatgtgcca caggtgtcct ccatgcagat gggagcagtg
     1141 gatctcctag gaggaggact agatagtctg cttggcagtg accttggcgg gggcattgga
     1201 ggaagtccgg cagtgggaca atccttcatc ccatcatcgg tccctgcaac ctttgctcct
     1261 tcacctacac ctgctgtggt cagcagtgga ctgaatgacc tgtttgaact ctccacaggg
     1321 ataggcatgg cacctggtgg atatgtggct cctaaggctg tctggctacc tgcagtaaag
     1381 gctaaaggct tggagatttc cggaacattt actcaccgcc aagggcacat ctatatggaa
     1441 atgaacttca ccaataaagc tctgcagcac atgacagatt ttgcaatcca gtttaacaaa
     1501 aatagctttg gtgtcatccc cagcactcct ctggccatcc atacaccact gatgccaaac
     1561 cagagcattg atgtctccct gcctctcaat accttgggcc cagtcatgaa gatggaacct
     1621 ctgaataacc tccaggtggc tgtgaaaaac aatatcgatg tcttcgcctt cagctgcctc
     1681 atcccactca atgtgctttt tgtagaagat ggcaaaatgg agcgccaggt cttccttgca
     1741 acatggaagg atattcccaa tgaaaatgaa cttcagtttc agattaagga atgtcattta
     1801 aatgctgaca ctgtttccag caagttgcaa aacaacaatg tttatactat tgccaagagg
     1861 aatgtggaag ggcaggacat gctgtaccaa tccctgaagc tcactaatgg catttggatt
     1921 ttggccgaac tacgtatcca gccaggaaac cccaattaca cgctgtcact gaagtgtaga
     1981 gctcctgaag tctctcaata catctatcag gtctacgaca gcattttgaa aaacttaccg
     2041 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2101 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2161 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2221 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2281 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2341 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2401 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2461 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2521 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2581 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2641 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2701 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2761 tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     2821 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2881 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2941 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3001 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3061 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3121 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3181 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3241 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3301 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3361 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3421 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3541 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3601 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3661 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3721 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3901 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3961 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4021 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4081 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4141 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4201 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4261 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4321 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4381 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4441 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4501 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4561 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4621 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4681 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4741 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4801 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4861 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4921 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4981 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     5041 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     5101 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     5161 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     5221 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5281 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5341 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5401 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5461 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5521 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5581 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5641 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5701 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5761 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5821 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5881 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5941 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6001 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6061 cgttatcccc tgattctgtg gataaccgta ttaccgccat
//
