LOCUS       sequence-193983-        5329 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 5329)
  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 5329)
  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-GTSE1(638-720)-GFP
FEATURES             Location/Qualifiers
     source          1..5329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        55..358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        359..562
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     513..533
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(660..662,663..983)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      1263..1272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1269..1271,1272..1274,1275..1988)
                     /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(1269..1271,1272..1274,1275..1985)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1314..1335)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1575..1594)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1922..1943
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2111..2232
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2148..2167)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2202..2221
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2239..2694)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2376..2397)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2588..2607
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2721..2825
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2827..3184
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2827..3018
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2850..2870)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3035..3170
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3219..4013
                     /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             3219..4013
                     /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(3273..3292)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3883..3902
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4201..4220)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4245..4292
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4621..5209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5110..5129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
       61 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      121 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      181 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      241 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      301 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      361 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      421 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      481 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      541 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggac
      601 tcagatctcg agagatcgat gtgaatgggg gccctgaacc cgccaagaac ctgtacacca
      661 tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc aagcgcggcc
      721 agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaattt gattcctccc
      781 gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc cgaggctggg
      841 aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt
      901 atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact ctcgtcttcg
      961 atgtggagct tctaaaactg gaacggtacc gcgggcccgg gatccacgat gctgcaagcc
     1021 agcccctcat tgaccttcct ctcatcgact tctgcgatac cccagaagca cacgtggctg
     1081 taggatctga aagcaggcct ctgatcgacc tcatgacaaa cactccagac atgaataaaa
     1141 atgtggccaa accttcaccg gtggtgggac agctcataga cctgagctcc cctctgatcc
     1201 agctgagccc tgaggctgac aaggagaacg tggattcccc actcctcaag ttcaatccgg
     1261 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1321 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1381 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1441 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1501 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1561 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1621 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1681 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1741 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1801 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1861 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1921 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1981 acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     2041 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2101 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2161 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2221 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2281 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2341 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2401 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2461 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2521 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2581 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2641 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     2701 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2761 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2821 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2881 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2941 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3001 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3061 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3121 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3181 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3241 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3301 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3361 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     3421 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3481 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3541 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3601 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3661 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3721 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     3781 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3841 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3901 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3961 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4021 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4081 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4141 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4201 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     4261 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     4321 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     4381 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     4441 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     4501 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4561 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4621 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4681 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4741 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4801 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4861 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4921 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4981 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5041 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5101 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5161 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5221 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5281 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagtta
//
