LOCUS       sequence-193573-        6073 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 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 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 Double
FEATURES             Location/Qualifiers
     source          1..6073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     525..545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(672..674,675..995)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      1020..1029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2019..2028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2025..2027,2028..2030,2031..2744)
                     /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(2025..2027,2028..2030,2031..2741)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2070..2091)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2331..2350)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2678..2699
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2867..2988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2904..2923)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2958..2977
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2995..3450)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3132..3153)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3344..3363
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3477..3581
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3583..3940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3583..3774
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3606..3626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3791..3926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3975..4769
                     /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             3975..4769
                     /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(4029..4048)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4639..4658
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4957..4976)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5001..5048
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5377..5965
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5866..5885
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      481 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      541 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      601 gcgctaccgg actcagatct cgagagatcg atgtgaatgg gggccctgaa cccgccaaga
      661 acctgtacac catgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc
      721 ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat
      781 ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga
      841 tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta
      901 tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca
      961 ctctcgtctt cgatgtggag cttctaaaac tggaacggta ccgcgggccc gggatccccg
     1021 ccaccatgga acagcctcag gttatcccct ctcaaggtga tctcggtccc ccagtcaatg
     1081 tgccacaggt gtcctccatg cagatgggag cagtggatct cctaggagga ggactagata
     1141 gtctgcttgg cagtgacctt ggcgggggca ttggaggaag tccggcagtg ggacaatcct
     1201 tcatcccatc atcggtccct gcaacctttg ctccttcacc tacacctgct gtggtcagca
     1261 gtggactgaa tgacctgttt gaactctcca cagggatagg catggcacct ggtggatatg
     1321 tggctcctaa ggctgtctgg ctacctgcag taaaggctaa aggcttggag atttccggaa
     1381 catttactca ccgccaaggg cacatctata tggaaatgaa cttcaccaat aaagctctgc
     1441 agcacatgac agattttgca atccagttta acaaaaatag ctttggtgtc atccccagca
     1501 ctcctctggc catccataca ccactgatgc caaaccagag cattgatgtc tccctgcctc
     1561 tcaatacctt gggcccagtc atgaagatgg aacctctgaa taacctccag gtggctgtga
     1621 aaaacaatat cgatgtcttc gccttcagct gcctcatccc actcaatgtg ctttttgtag
     1681 aagatggcaa aatggagcgc caggtcttcc ttgcaacatg gaaggatatt cccaatgaaa
     1741 atgaacttca gtttcagatt aaggaatgtc atttaaatgc tgacactgtt tccagcaagt
     1801 tgcaaaacaa caatgtttat actattgcca agaggaatgt ggaagggcag gacatgctgt
     1861 accaatccct gaagctcact aatggcattt ggattttggc cgaactacgt atccagccag
     1921 gaaaccccaa ttacacgctg tcactgaagt gtagagctcc tgaagtctct caatacatct
     1981 atcaggtcta cgacagcatt ttgaaaaact taccggtcgc caccatggtg agcaagggcg
     2041 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     2101 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     2161 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     2221 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     2281 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2341 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2401 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2461 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     2521 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2581 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2641 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2701 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta
     2761 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     2821 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     2881 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2941 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     3001 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     3061 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     3121 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     3181 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     3241 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3301 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     3361 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     3421 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     3481 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3541 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     3601 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3661 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3721 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3781 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3841 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3901 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     3961 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4021 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4081 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4141 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4201 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4261 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4321 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4381 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4441 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4501 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4561 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4621 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4681 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4741 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4801 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4861 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4921 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     4981 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     5041 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     5101 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     5161 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     5221 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5281 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5341 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5401 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5461 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5521 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5581 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5641 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5701 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5761 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5821 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5881 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5941 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6001 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6061 ccgtattacc gcc
//
