LOCUS       sequence-193984-        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        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1218..1227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2244..2253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2250..2960
                     /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(2395..2413)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2663..2682
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3082..3203
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3119..3138)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3173..3192
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3210..3665)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3347..3368)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3559..3578
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3692..3796
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3798..4155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3798..3989
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3821..3841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4006..4141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4190..4984
                     /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             4190..4984
                     /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(4244..4263)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4854..4873
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5172..5191)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5216..5263
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5592..6180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6081..6100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggactc agatctcgag gccaccatgc cagaacttgg atttagcata tcaggtggtg
      661 tcgggggtag aggaaaccca ttcagacctg atgatgatgg tatatttgta acaagggtac
      721 aacctgaagg accagcatca aaattactgc agccaggtga taaaattatt caggctaatg
      781 gctacagttt tataaatatt gaacatggac aagcagtgtc cttgctaaaa actttccaga
      841 atacagttga actcatcatt gtacgagaag ttggtaacgg tgcaaaacaa gagattcgag
      901 tgagggttga aaaagacggt ggttctggag gcgtttcttc tgttccgacc aatctggaag
      961 ttgttgctgc gaccccgact agcctgctga tcagctggga tgcgtactac gactctcatg
     1021 tttcttatta ccgtatcacg tacggtgaaa ccggtggtaa ctccccggtt caggaattca
     1081 ctgtacctgg ttccaagtct actgctacca tcagcggcct gaaaccgggt gtcgactata
     1141 ccatcactgt atacgcacat tacaactacc attactactc tagcccaatc tcgattaact
     1201 accgtaccag gatccccgcc accatggaac agcctcaggt tatcccctct caaggtgatc
     1261 ttctagggga tcttttaaac cttgacctcg gtcccccagt caatgtgcca caggtgtcct
     1321 ccatgcagat gggagcagtg gatctcctag gaggaggact agatagtctg cttggcagtg
     1381 accttggcgg gggcattgga ggaagtccgg cagtgggaca atccttcatc ccatcatcgg
     1441 tccctgcaac ctttgctcct tcacctacac ctgctgtggt cagcagtgga ctgaatgacc
     1501 tgtttgaact ctccacaggg ataggcatgg cacctggtgg atatgtggct cctaaggctg
     1561 tctggctacc tgcagtaaag gctaaaggct tggagatttc cggaacattt actcaccgcc
     1621 aagggcacat ctatatggaa atgaacttca ccaataaagc tctgcagcac atgacagatt
     1681 ttgcaatcca gtttaacaaa aatagctttg gtgtcatccc cagcactcct ctggccatcc
     1741 atacaccact gatgccaaac cagagcattg atgtctccct gcctctcaat accttgggcc
     1801 cagtcatgaa gatggaacct ctgaataacc tccaggtggc tgtgaaaaac aatatcgatg
     1861 tcttctactt cagctgcctc atcccactca atgtgctttt tgtagaagat ggcaaaatgg
     1921 agcgccaggt cttccttgca acatggaagg atattcccaa tgaaaatgaa cttcagtttc
     1981 agattaagga atgtcattta aatgctgaca ctgtttccag caagttgcaa aacaacaatg
     2041 tttatactat tgccaagagg aatgtggaag ggcaggacat gctgtaccaa tccctgaagc
     2101 tcactaatgg catttggatt ttggccgaac tacgtatcca gccaggaaac cccaattaca
     2161 cgctgtcact gaagtgtaga gctcctgaag tctctcaata catctatcag gtctacgaca
     2221 gcattttgaa aaacttaccg gtcgccacca tggtgagcaa gggcgaggag gataacatgg
     2281 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     2341 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     2401 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     2461 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     2521 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2581 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2641 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     2701 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2761 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2821 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     2881 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2941 tggacgagct gtacaagtaa gcggccgcga ctctagatca taatcagcca taccacattt
     3001 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     3061 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     3121 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3181 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     3241 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3301 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3361 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3421 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3481 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3541 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3601 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3661 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3721 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3781 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3901 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3961 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4021 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4081 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4141 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4201 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4261 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4321 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4381 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4441 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4501 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4561 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4621 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4681 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4741 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4801 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4861 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4921 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4981 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5041 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5101 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     5161 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     5221 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     5281 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     5341 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5401 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5461 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5521 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5581 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5641 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5701 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5761 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5821 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5881 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5941 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6001 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6061 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6121 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6181 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6241 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gc
//
