LOCUS       sequence-189378-        6292 bp    DNA     circular SYN 15-DEC-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 6292)
  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 6292)
  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: PDZb-beta2(616-951)-mCherry
FEATURES             Location/Qualifiers
     source          1..6292
                     /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"
     regulatory      1216..1225
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2242..2251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2248..2958
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2393..2411)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2661..2680
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3080..3201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3117..3136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3171..3190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3208..3663)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3345..3366)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3557..3576
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3690..3794
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3796..4153
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3796..3987
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3819..3839)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4004..4139
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4188..4982
                     /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             4188..4982
                     /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(4242..4261)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4852..4871
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5170..5189)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5214..5261
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5590..6178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6079..6098
                     /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 atctcgaggc caccatgcca gaacttggat ttagcatatc aggtggtgtc
      661 gggggtagag gaaacccatt cagacctgat gatgatggta tatttgtaac aagggtacaa
      721 cctgaaggac cagcatcaaa attactgcag ccaggtgata aaattattca ggctaatggc
      781 tacagtttta taaatattga acatggacaa gcagtgtcct tgctaaaaac tttccagaat
      841 acagttgaac tcatcattgt acgagaagtt ggtaacggtg caaaacaaga gattcgagtg
      901 agggttgaaa aagacggtgg ttctggaggc gtttcttctg ttccgaccaa tctggaagtt
      961 gttgctgcga ccccgactag cctgctgatc agctgggatg cgtactacga ctctcatgtt
     1021 tcttattacc gtatcacgta cggtgaaacc ggtggtaact ccccggttca ggaattcact
     1081 gtacctggtt ccaagtctac tgctaccatc agcggcctga aaccgggtgt cgactatacc
     1141 atcactgtat acgcacatta caactaccat tactactcta gcccaatctc gattaactac
     1201 cgtaccagga tccccgccac catggaacag cctcaggtta tcccctctca aggtgatctt
     1261 ctaggggatc ttttaaacct tgacctcggt cccccagtca atgtgccaca ggtgtcctcc
     1321 atgcagatgg gagcagtgga tctcctagga ggaggactag atagtctgct tggcagtgac
     1381 cttggcgggg gcattggagg aagtccggca gtgggacaat ccttcatccc atcatcggtc
     1441 cctgcaacct ttgctccttc acctacacct gctgtggtca gcagtggact gaatgacctg
     1501 tttgaactct ccacagggat aggcatggca cctggtggat atgtggctcc taaggctgtc
     1561 tggctacctg cagtaaaggc taaaggcttg gagatttccg gaacatttac tcaccgccaa
     1621 gggcacatct atatggaaat gaacttcacc aataaagctc tgcagcacat gacagatttt
     1681 gcaatccagt ttaacaaaaa tagctttggt gtcatcccca gcactcctct ggccatccat
     1741 acaccactga tgccaaacca gagcattgat gtctccctgc ctctcaatac cttgggccca
     1801 gtcatgaaga tggaacctct gaataacctc caggtggctg tgaaaaacaa tatcgatgtc
     1861 ttctacttca gctgcctcat cccactcaat gtgctttttg tagaagatgg caaaatggag
     1921 cgccaggtct tccttgcaac atggaaggat attcccaatg aaaatgaact tcagtttcag
     1981 attaaggaat gtcatttaaa tgctgacact gtttccagca agttgcaaaa caacaatgtt
     2041 tatactattg ccaagaggaa tgtggaaggg caggacatgc tgtaccaatc cctgaagctc
     2101 actaatggca tttggatttt ggccgaacta cgtatccagc caggaaaccc caattacacg
     2161 ctgtcactga agtgtagagc tcctgaagtc tctcaataca tctatcaggt ctacgacagc
     2221 attttgaaaa acttaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc
     2281 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     2341 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     2401 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     2461 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     2521 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     2581 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     2641 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     2701 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     2761 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     2821 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     2881 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     2941 gacgagctgt acaagtaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     3001 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3061 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3121 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3181 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     3241 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     3301 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3361 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3421 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3481 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3541 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3601 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3661 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3721 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3781 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     3841 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3901 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3961 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4021 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4081 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4141 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4201 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4261 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4321 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     4381 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4441 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4501 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4561 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4621 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4681 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4741 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4801 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4861 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4921 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4981 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5041 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5101 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     5161 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     5221 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     5281 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     5341 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     5401 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     5461 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5521 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5581 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5641 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5701 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     5761 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5821 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5881 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5941 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6001 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6061 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6121 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6181 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6241 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
