LOCUS       sequence-189369-        6100 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 6100)
  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 6100)
  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 Y215A
FEATURES             Location/Qualifiers
     source          1..6100
                     /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      2040..2049
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2046..2048,2049..2051,2052..2765)
                     /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(2046..2048,2049..2051,2052..2762)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2091..2112)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2352..2371)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2699..2720
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2888..3009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2925..2944)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2979..2998
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3016..3471)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3153..3174)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3365..3384
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3498..3602
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3604..3961
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3604..3795
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3627..3647)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3812..3947
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3996..4790
                     /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             3996..4790
                     /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(4050..4069)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4660..4679
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4978..4997)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5022..5069
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5398..5986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5887..5906
                     /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 gtgatcttct aggggatctt ttaaaccttg
     1081 acctcggtcc cccagtcaat gtgccacagg tgtcctccat gcagatggga gcagtggatc
     1141 tcctaggagg aggactagat agtctgcttg gcagtgacct tggcgggggc attggaggaa
     1201 gtccggcagt gggacaatcc ttcatcccat catcggtccc tgcaaccttt gctccttcac
     1261 ctacacctgc tgtggtcagc agtggactga atgacctgtt tgaactctcc acagggatag
     1321 gcatggcacc tggtggatat gtggctccta aggctgtctg gctacctgca gtaaaggcta
     1381 aaggcttgga gatttccgga acatttactc accgccaagg gcacatctat atggaaatga
     1441 acttcaccaa taaagctctg cagcacatga cagattttgc aatccagttt aacaaaaata
     1501 gctttggtgt catccccagc actcctctgg ccatccatac accactgatg ccaaaccaga
     1561 gcattgatgt ctccctgcct ctcaatacct tgggcccagt catgaagatg gaacctctga
     1621 ataacctcca ggtggctgtg aaaaacaata tcgatgtctt cgccttcagc tgcctcatcc
     1681 cactcaatgt gctttttgta gaagatggca aaatggagcg ccaggtcttc cttgcaacat
     1741 ggaaggatat tcccaatgaa aatgaacttc agtttcagat taaggaatgt catttaaatg
     1801 ctgacactgt ttccagcaag ttgcaaaaca acaatgttta tactattgcc aagaggaatg
     1861 tggaagggca ggacatgctg taccaatccc tgaagctcac taatggcatt tggattttgg
     1921 ccgaactacg tatccagcca ggaaacccca attacacgct gtcactgaag tgtagagctc
     1981 ctgaagtctc tcaatacatc tatcaggtct acgacagcat tttgaaaaac ttaccggtcg
     2041 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2101 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2161 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2221 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2281 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2341 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2401 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2461 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2521 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2581 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2641 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2701 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2761 agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2821 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2881 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2941 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3001 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     3061 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     3121 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     3181 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     3241 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3301 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3361 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3421 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     3481 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3601 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3661 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3721 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3781 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3841 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3901 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3961 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4021 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4081 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4141 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     4201 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4261 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4321 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4381 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4441 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4501 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     4561 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4621 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4681 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4741 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4801 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4861 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4921 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4981 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     5041 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     5101 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     5161 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     5221 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     5281 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5341 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5401 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5461 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5521 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5581 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5641 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5701 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5761 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5821 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5881 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5941 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6001 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6061 atcccctgat tctgtggata accgtattac cgccatgcat
//
