LOCUS       sequence-194007-        5061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a plasma membrane ""anchor"" in mammalian cells to 
            hot-wire endocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5061)
  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 5061)
  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: SH4-FRB-mRFP
FEATURES             Location/Qualifiers
     source          1..5061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        363..566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     517..537
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             complement(674..691)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             706..984
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             991..1017
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1024..1033
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1030..1704
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             1030..1704
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(1160..1178)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1428..1447
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1622..1645
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1847..1968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1884..1903)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1938..1957
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1975..2430)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2133)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2324..2343
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2457..2561
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2563..2920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2563..2754
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2586..2606)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2771..2906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2955..3749
                     /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             2955..3749
                     /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(3009..3028)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3619..3638
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3937..3956)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3981..4028
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4357..4945
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4846..4865
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
       61 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      121 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      181 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      241 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      301 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      361 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      421 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      481 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      541 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcaccatg
      601 ggctgtgtgc aatgtaagga taaagaagca acaaaactga cggaggatcc caccagatct
      661 gcgaattcag gtgctggtgc tggtgctggt gctatcctat ctagaatcct ctggcatgag
      721 atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa
      781 ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg
      841 aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc
      901 aggaagtaca tgaaatcagg gaatgtcaag gacctcaccc aagcctggga cctctattat
      961 catgtgttcc gacgaatctc aaagactagt tatccgtacg acgtaccaga ctacgcaccg
     1021 gtcgccacca tggcctcctc cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc
     1081 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1141 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc
     1201 tgggacatcc tgtcccctca gttccagtac ggctccaagg cctacgtgaa gcaccccgcc
     1261 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1321 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1381 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1441 aagaagacca tgggctggga ggcctccacc gagcggatgt accccgagga cggcgccctg
     1501 aagggcgaga tcaagatgag gctgaagctg aaggacggcg gccactacga cgccgaggtc
     1561 aagaccacct acatggccaa gaagcccgtg cagctgcccg gcgcctacaa gaccgacatc
     1621 aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgcgcc
     1681 gagggccgcc actccaccgg cgcctccgga ctcagatctc gaagcgcggc cgcgactcta
     1741 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     1801 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     1861 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1921 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     1981 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2041 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2101 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2161 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2221 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2281 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2341 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2401 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     2461 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2521 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     2581 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2641 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2701 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2761 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2821 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2881 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     2941 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3001 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3061 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3121 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3181 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3241 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3301 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3361 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3421 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3481 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3541 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3601 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3661 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3721 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3781 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3841 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3901 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     3961 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4021 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4081 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4141 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4201 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4261 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4321 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4381 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4441 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4501 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4561 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4621 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4681 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4741 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4801 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4861 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4921 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4981 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5041 ccgtattacc gccatgcatt a
//
