LOCUS       sequence-189380-        5757 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 5757)
  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 5757)
  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: CD8-mCherry-FRB
FEATURES             Location/Qualifiers
     source          1..5757
                     /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      1382..1391
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1388..2092
                     /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
                     EGRHSTGGMDELY"
     primer_bind     complement(1533..1551)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1801..1820
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2096..2122
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2141..2419
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2153..2422
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    2545..2666
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2582..2601)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2636..2655
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2673..3128)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2810..2831)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3022..3041
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3155..3259
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3261..3618
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3261..3452
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3284..3304)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3469..3604
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3653..4447
                     /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             3653..4447
                     /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(3707..3726)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4317..4336
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4635..4654)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4679..4726
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5055..5643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5544..5563
                     /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 attcggcggc tcccgcgcgc ctcccctcgc
      661 gcccgagctt cgagccaagc agcgtcctgg ggagcgcgtc atggccttac cagtgaccgc
      721 cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccgagccagt tccgggtgtc
      781 gccgctggat cggacctgga acctgggcga gacagtggag ctgaagtgcc aggtgctgct
      841 gtccaacccg acgtcgggct gctcgtggct cttccagccg cgcggcgccg ccgccagtcc
      901 caccttcctc ctatacctct cccaaaacaa gcccaaggcg gccgaggggc tggacaccca
      961 gcggttctcg ggcaagaggt tgggggacac cttcgtcctc accctgagcg acttccgccg
     1021 agagaacgag ggctactatt tctgctcggc cctgagcaac tccatcatgt acttcagcca
     1081 cttcgtgccg gtcttcctgc cagcgaagcc caccacgacg ccagcgccgc gaccaccaac
     1141 accggcgccc accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc
     1201 ggcggggggc gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc
     1261 gcccttggcc gggacttgtg gagtccttct cctgtcactg gttatcaccc tttactgcaa
     1321 gcggcttaag cgaagaagga tccccgccgc cgccgccgcc gccgccgccg tcagaccggt
     1381 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     1441 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     1501 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     1561 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     1621 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     1681 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     1741 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     1801 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     1861 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     1921 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     1981 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     2041 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acacttaccc
     2101 ctacgacgtg cccgactacg caggctccgc acgagtggcc atcctctggc atgagatgtg
     2161 gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg tgaaaggcat
     2221 gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga ctctgaagga
     2281 aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt ggtgcaggaa
     2341 gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct attatcatgt
     2401 gttccgacga atctcaaagc aggccggctg ataatctaga tcataatcag ccataccaca
     2461 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     2521 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     2581 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2641 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
     2701 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2761 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2821 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2881 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2941 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     3001 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     3061 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3121 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3181 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3241 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     3301 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3361 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3421 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3481 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3541 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3601 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     3661 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3721 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3781 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     3841 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3901 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3961 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4021 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4081 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4141 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     4201 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4261 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4321 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4381 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4441 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4501 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4561 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     4621 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     4681 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     4741 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     4801 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     4861 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     4921 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4981 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5041 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5101 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5161 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5221 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5281 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5341 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5401 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5461 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5521 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5581 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5641 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5701 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat
//
