LOCUS       sequence-193577-        6060 bp    DNA     circular SYN 14-APR-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 6060)
  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 6060)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-beta1(617-949)-GFP
FEATURES             Location/Qualifiers
     source          1..6060
                     /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(635..637,638..958)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2000..2009
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2006..2008,2009..2011,2012..2725)
                     /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(2006..2008,2009..2011,2012..2722)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2051..2072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2312..2331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2659..2680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2848..2969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2885..2904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2939..2958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2976..3431)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3113..3134)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3325..3344
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3458..3562
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3564..3921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3564..3755
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3587..3607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3772..3907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3956..4750
                     /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             3956..4750
                     /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(4010..4029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4620..4639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4938..4957)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4982..5029
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5358..5946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5847..5866
                     /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 atctcgagct caagcttcga attcatggga gtgcaggtgg aaaccatctc
      661 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg
      721 gatgcttgaa gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt
      781 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt
      841 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc
      901 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaacg
      961 gtaccgcggg cccgggttcc ccgccaccat ggagcagcca gatgtcatcc ccgcccaggg
     1021 cgacctgctg ggtgacctcc tcaacctgga cctcggcccc ccagtgagcg gcccacccct
     1081 ggccacctcc tcggtgcaga tgggagctgt ggaccttctt ggcggtggcc ttgacagcct
     1141 gatgggggat gagcctgaag ggattggggg caccaacttc gtggcacctc caacagcagc
     1201 agtaccagcc aatcttggag cacccatcgg cagtggcctg agtgacctct ttgacctgac
     1261 cagtggcgtg ggcaccctgt caggatcata tgtggccccc aaagcagtct ggctcccagc
     1321 catgaaggct aaggggctgg agatctcagg caccttcacc cgccaggtgg gctccatctc
     1381 catggacctg cagctgacca acaaggcctt gcaggtcatg accgactttg ccatccagtt
     1441 caaccgcaac agctttggcc tggcccccgc cgcccccctc caggtccacg cgccactcag
     1501 ccccaaccag acagtggaga tctccctgcc tctcagcacg gtgggctcgg tcatgaagat
     1561 ggagcctctg aacaacctcc aggtggccgt gaagaacaac atcgatgtct tctacttcag
     1621 caccttgtac ccactgcaca tcctctttgt ggaggacggg aagatggacc ggcagatgtt
     1681 cctggccaca tggaaggata ttcccaatga gaatgaggcc cagttccaga tcagagactg
     1741 ccccctcaat gcagaggctg cgagcagcaa gctgcagagc agcaacatct tcactgtcgc
     1801 caagaggaac gtggagggcc aggacatgct ctaccagtcc ctgaagctga ccaacggcat
     1861 ctgggtgctg gcggagctgc ggatccagcc gggcaacccc agctgcacgg acttagagct
     1921 gtccctgaag tgtcgagcac cagaggtgtc ccagcacgtg taccaggcct acgagaccat
     1981 cctcaagaac ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2041 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2101 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2161 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2221 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2281 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2341 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2401 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2461 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2521 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2581 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2641 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2701 cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc
     2761 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2821 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2881 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2941 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     3001 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3061 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3121 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3181 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3241 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3301 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3361 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3421 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3481 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3541 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3601 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3661 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3721 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3781 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3841 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3901 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3961 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4021 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4081 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4141 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4201 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4261 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4321 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4381 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4441 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4501 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4561 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4621 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4681 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4741 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4801 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4861 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4921 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4981 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5041 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5101 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5161 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5221 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5281 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5341 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5401 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5461 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5521 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5581 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5641 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5701 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5761 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5821 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5881 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5941 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6001 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
