LOCUS       sequence-189370-        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 ∆CBM
FEATURES             Location/Qualifiers
     source          1..6073
                     /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      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2013..2022
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2019..2021,2022..2024,2025..2738)
                     /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(2019..2021,2022..2024,2025..2735)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2064..2085)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2325..2344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2672..2693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2861..2982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2898..2917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2952..2971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2989..3444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3338..3357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3471..3575
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3577..3934
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3577..3768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3600..3620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3785..3920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3969..4763
                     /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             3969..4763
                     /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(4023..4042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4633..4652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4951..4970)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4995..5042
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5371..5959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5860..5879
                     /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 ggtaccgcgg gcccgggatc cccgccacca
     1021 tggaacagcc tcaggttatc ccctctcaag gtgatctcgg tcccccagtc aatgtgccac
     1081 aggtgtcctc catgcagatg ggagcagtgg atctcctagg aggaggacta gatagtctgc
     1141 ttggcagtga ccttggcggg ggcattggag gaagtccggc agtgggacaa tccttcatcc
     1201 catcatcggt ccctgcaacc tttgctcctt cacctacacc tgctgtggtc agcagtggac
     1261 tgaatgacct gtttgaactc tccacaggga taggcatggc acctggtgga tatgtggctc
     1321 ctaaggctgt ctggctacct gcagtaaagg ctaaaggctt ggagatttcc ggaacattta
     1381 ctcaccgcca agggcacatc tatatggaaa tgaacttcac caataaagct ctgcagcaca
     1441 tgacagattt tgcaatccag tttaacaaaa atagctttgg tgtcatcccc agcactcctc
     1501 tggccatcca tacaccactg atgccaaacc agagcattga tgtctccctg cctctcaata
     1561 ccttgggccc agtcatgaag atggaacctc tgaataacct ccaggtggct gtgaaaaaca
     1621 atatcgatgt cttctacttc agctgcctca tcccactcaa tgtgcttttt gtagaagatg
     1681 gcaaaatgga gcgccaggtc ttccttgcaa catggaagga tattcccaat gaaaatgaac
     1741 ttcagtttca gattaaggaa tgtcatttaa atgctgacac tgtttccagc aagttgcaaa
     1801 acaacaatgt ttatactatt gccaagagga atgtggaagg gcaggacatg ctgtaccaat
     1861 ccctgaagct cactaatggc atttggattt tggccgaact acgtatccag ccaggaaacc
     1921 ccaattacac gctgtcactg aagtgtagag ctcctgaagt ctctcaatac atctatcagg
     1981 tctacgacag cattttgaaa aacttaccgg tcgccaccat ggtgagcaag ggcgaggagc
     2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2101 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2161 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2221 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2281 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2461 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2521 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2581 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2641 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2701 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat
     2761 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2821 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2881 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2941 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     3001 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3061 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3121 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3181 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3241 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3301 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3361 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3421 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3541 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3601 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3661 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3721 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3781 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3841 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3901 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     3961 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4021 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4081 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4141 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4201 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4261 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4321 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4381 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4441 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4501 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4561 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4621 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4681 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4741 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4921 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     4981 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     5041 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     5101 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     5161 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     5221 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5281 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5341 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5401 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5461 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5521 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5581 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5641 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5701 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5761 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5821 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5881 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5941 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6001 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6061 taccgccatg cat
//
