LOCUS       sequence-193576-        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        57..360
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        361..564
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     515..535
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(662..664,665..985)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             complement(1352..1363)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     regulatory      1730..1739
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1736..1738,1739..1741,1742..2455)
                     /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(1736..1738,1739..1741,1742..2452)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1781..1802)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2042..2061)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2389..2410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2578..2699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2615..2634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2669..2688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2706..3161)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2843..2864)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3055..3074
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3188..3292
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3294..3651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3294..3485
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3317..3337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3502..3637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3686..4480
                     /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             3686..4480
                     /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(3740..3759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4350..4369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4668..4687)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4712..4759
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5088..5676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5577..5596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
       61 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      121 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      181 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      241 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      301 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      361 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      421 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      481 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      541 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      601 actcagatct cgagagatcg atgtgaatgg gggccctgaa cccgccaaga acctgtacac
      661 catgggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg
      721 ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaat ttgattcctc
      781 ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga tccgaggctg
      841 ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta tatctccaga
      901 ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca ctctcgtctt
      961 cgatgtggag cttctaaaac tggaacggta ccgcggtcca ggtatcgcag atgaactgtt
     1021 gaataagttc gtgtgtaaga atagtggggt cttgtttgag aaccagctgc tgcagattgg
     1081 agtcaagtct gagttccggc agaacctggg ccgcatgtat ctcttctatg gcaacaagac
     1141 ttctgtgcag ttccagaact tcttgcccac cgtggtccat cctggggacc tccagactca
     1201 gctggcggtg cagaccaagc gtgtggcggc acaagtggac ggtggcgcac aggtgcagca
     1261 agtactcaac attgagtgtc tgcgagactt cctgacgccg ccactgttgt cggtgcgctt
     1321 ccggtacggt ggcaccgccc agtccctcac tctgaagctc ccagtgacca tcaacaaatt
     1381 cttccagccc acagagatgg cggcccaaga ctttttccag cgctggaagc agctgagcct
     1441 ccccctgcag gaggcacaga aaatcttcaa agccaaccac ccgatggatg ctgaagttac
     1501 taaggccaag cttctggggt ttggctctgc tcttctggac aatgtggatc ccaaccctga
     1561 gaactttgtg ggtgctggaa tcatccagac gaaggccctg caggtggggt gtctgcttcg
     1621 gctggagccc aatgcccagg cccaaatgta ccgtctaacc ctgcgcacca gcaaagagcc
     1681 tgtctcccgt cacttgtgtg agctgctggc ccagcagttc caaccggtcg ccaccatggt
     1741 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1801 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1861 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1921 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1981 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2041 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2101 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2161 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2221 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2281 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2341 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2401 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     2461 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2521 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2701 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2761 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2821 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2881 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2941 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3001 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3061 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3121 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     3181 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3241 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     3301 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3361 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3421 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3481 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3541 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3601 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3661 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3721 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3781 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3841 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3901 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3961 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4021 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4081 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4141 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4201 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4261 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4321 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4381 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4441 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4501 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4561 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4621 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4681 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4741 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4801 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4861 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4921 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4981 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5041 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5101 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5161 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5221 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5281 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5341 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5401 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5461 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5521 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5581 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5641 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5701 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5761 tctgtggata accgtattac cgccatgcat tagt
//
