LOCUS       sequence-239632-        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: PDZb1-beta2(616-951)-mCherry
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        423..626
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     577..597
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1274..1283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2300..2309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2306..3016
                     /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(2451..2469)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2719..2738
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3138..3259
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3175..3194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3229..3248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3266..3721)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3403..3424)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3615..3634
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3748..3852
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3854..4211
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3854..4045
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3877..3897)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4062..4197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4246..5040
                     /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             4246..5040
                     /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(4300..4319)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4910..4929
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5228..5247)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5272..5319
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5648..6236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6137..6156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
       61 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      481 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      541 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      601 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      661 ggactcagat ctcgaggcca ccatgccaga acttggattt agcatatcag gtggtgtcgg
      721 gggtagagga aacccattca gacctgatga tgatggtata tttgtaacaa gggtacaacc
      781 tgaaggacca gcatcaaaat tactgcagcc aggtgataaa attattcagg ctaatggcta
      841 cagttttata aatattgaac atggacaagc agtgtccttg ctaaaaactt tccagaatac
      901 agttgaactc atcattgtac gagaagttgg taacggtgca aaacaagaga ttcgagtgag
      961 ggttgaaaaa gacggtggtt ctggaggcgt ttcttctgtt ccgaccaatc tggaagttgt
     1021 tgctgcgacc ccgactagcc tgctgatcag ctgggatgcg tatagggagc ttccggttag
     1081 ttattaccgt atcacgtacg gtgaaaccgg tggtaactcc ccggttcagg aattcactgt
     1141 acctggttcc aagtctactg ctaccatcag cggcctgaaa ccgggtgtcg actataccat
     1201 cactgtatac gcacattaca actaccatta ctactctagc ccaatctcga ttaactaccg
     1261 taccaggatc cccgccacca tggaacagcc tcaggttatc ccctctcaag gtgatcttct
     1321 aggggatctt ttaaaccttg acctcggtcc cccagtcaat gtgccacagg tgtcctccat
     1381 gcagatggga gcagtggatc tcctaggagg aggactagat agtctgcttg gcagtgacct
     1441 tggcgggggc attggaggaa gtccggcagt gggacaatcc ttcatcccat catcggtccc
     1501 tgcaaccttt gctccttcac ctacacctgc tgtggtcagc agtggactga atgacctgtt
     1561 tgaactctcc acagggatag gcatggcacc tggtggatat gtggctccta aggctgtctg
     1621 gctacctgca gtaaaggcta aaggcttgga gatttccgga acatttactc accgccaagg
     1681 gcacatctat atggaaatga acttcaccaa taaagctctg cagcacatga cagattttgc
     1741 aatccagttt aacaaaaata gctttggtgt catccccagc actcctctgg ccatccatac
     1801 accactgatg ccaaaccaga gcattgatgt ctccctgcct ctcaatacct tgggcccagt
     1861 catgaagatg gaacctctga ataacctcca ggtggctgtg aaaaacaata tcgatgtctt
     1921 ctacttcagc tgcctcatcc cactcaatgt gctttttgta gaagatggca aaatggagcg
     1981 ccaggtcttc cttgcaacat ggaaggatat tcccaatgaa aatgaacttc agtttcagat
     2041 taaggaatgt catttaaatg ctgacactgt ttccagcaag ttgcaaaaca acaatgttta
     2101 tactattgcc aagaggaatg tggaagggca ggacatgctg taccaatccc tgaagctcac
     2161 taatggcatt tggattttgg ccgaactacg tatccagcca ggaaacccca attacacgct
     2221 gtcactgaag tgtagagctc ctgaagtctc tcaatacatc tatcaggtct acgacagcat
     2281 tttgaaaaac ttaccggtcg ccaccatggt gagcaagggc gaggaggata acatggccat
     2341 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     2401 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     2461 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     2521 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     2581 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     2641 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     2701 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc
     2761 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     2821 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     2881 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     2941 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     3001 cgagctgtac aagtaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     3061 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3121 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3181 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3241 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     3301 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3361 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3421 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3481 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3541 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3601 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3661 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3721 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3781 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3841 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3901 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3961 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4021 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4081 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4141 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4201 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4261 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4321 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4381 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4441 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4501 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4561 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4621 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4681 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4741 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4801 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4861 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4921 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4981 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5041 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5101 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5161 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     5221 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5281 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     5341 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     5401 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     5461 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     5521 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5581 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5641 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5701 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5761 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5821 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5881 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5941 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6001 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6061 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6121 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6181 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6241 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct ca
//
