LOCUS       sequence-189373-        5794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a clathrin ""hook"" in mammalian cells to hot-wire 
            endocytosis (negative control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5794)
  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 5794)
  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-alpha(740-977)-GFP
FEATURES             Location/Qualifiers
     source          1..5794
                     /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"
     CDS             complement(1356..1367)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     regulatory      1734..1743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1740..1742,1743..1745,1746..2459)
                     /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(1740..1742,1743..1745,1746..2456)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1785..1806)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2046..2065)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2393..2414
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2582..2703
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2619..2638)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2673..2692
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2710..3165)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2847..2868)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3059..3078
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3192..3296
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3298..3655
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3298..3489
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3321..3341)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3506..3641
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3690..4484
                     /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             3690..4484
                     /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(3744..3763)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4354..4373
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4672..4691)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4716..4763
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5092..5680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5581..5600
                     /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 tccaggtatc gcagatgaac
     1021 tgttgaataa gttcgtgtgt aagaatagtg gggtcttgtt tgagaaccag ctgctgcaga
     1081 ttggagtcaa gtctgagttc cggcagaacc tgggccgcat gtatctcttc tatggcaaca
     1141 agacttctgt gcagttccag aacttcttgc ccaccgtggt ccatcctggg gacctccaga
     1201 ctcagctggc ggtgcagacc aagcgtgtgg cggcacaagt ggacggtggc gcacaggtgc
     1261 agcaagtact caacattgag tgtctgcgag acttcctgac gccgccactg ttgtcggtgc
     1321 gcttccggta cggtggcacc gcccagtccc tcactctgaa gctcccagtg accatcaaca
     1381 aattcttcca gcccacagag atggcggccc aagacttttt ccagcgctgg aagcagctga
     1441 gcctccccct gcaggaggca cagaaaatct tcaaagccaa ccacccgatg gatgctgaag
     1501 ttactaaggc caagcttctg gggtttggct ctgctcttct ggacaatgtg gatcccaacc
     1561 ctgagaactt tgtgggtgct ggaatcatcc agacgaaggc cctgcaggtg gggtgtctgc
     1621 ttcggctgga gcccaatgcc caggcccaaa tgtaccgtct aaccctgcgc accagcaaag
     1681 agcctgtctc ccgtcacttg tgtgagctgc tggcccagca gttccaaccg gtcgccacca
     1741 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1801 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1861 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1921 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1981 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2041 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2101 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2161 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2221 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2281 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2341 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2401 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     2461 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2521 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2581 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2641 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2701 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2761 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2821 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2881 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2941 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3001 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3061 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3121 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     3301 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3361 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3421 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3481 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3541 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3601 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3661 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3721 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3781 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3841 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3901 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3961 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4021 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4081 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4141 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4201 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4261 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4321 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4381 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4441 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4501 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4561 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4621 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4681 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4741 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     4801 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     4861 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     4921 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     4981 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5041 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5101 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5161 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5221 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5281 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5341 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5401 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5461 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5521 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5581 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5641 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5701 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5761 tgattctgtg gataaccgta ttaccgccat gcat
//
