LOCUS       sequence-193991-        5757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a plasma membrane ""anchor"" in mammalian cells to 
            hot-wire endocytosis (dark mCherry K70N).
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-dCherry-FRB
FEATURES             Location/Qualifiers
     source          1..5757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        57..360
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        361..564
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     515..535
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1378..1387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1384..2088
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSNAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(1529..1547)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1797..1816
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2092..2118
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2137..2415
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2149..2418
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    2541..2662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2578..2597)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2632..2651
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2669..3124)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2806..2827)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3018..3037
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3151..3255
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3257..3614
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3257..3448
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3280..3300)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3465..3600
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3649..4443
                     /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             3649..4443
                     /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(3703..3722)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4313..4332
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4631..4650)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4675..4722
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5051..5639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5540..5559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
       61 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      121 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      181 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      241 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      301 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      361 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      421 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      481 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      541 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      601 actcagatct cgagctcaag cttcgaattc ggcggctccc gcgcgcctcc cctcgcgccc
      661 gagcttcgag ccaagcagcg tcctggggag cgcgtcatgg ccttaccagt gaccgccttg
      721 ctcctgccgc tggccttgct gctccacgcc gccaggccga gccagttccg ggtgtcgccg
      781 ctggatcgga cctggaacct gggcgagaca gtggagctga agtgccaggt gctgctgtcc
      841 aacccgacgt cgggctgctc gtggctcttc cagccgcgcg gcgccgccgc cagtcccacc
      901 ttcctcctat acctctccca aaacaagccc aaggcggccg aggggctgga cacccagcgg
      961 ttctcgggca agaggttggg ggacaccttc gtcctcaccc tgagcgactt ccgccgagag
     1021 aacgagggct actatttctg ctcggccctg agcaactcca tcatgtactt cagccacttc
     1081 gtgccggtct tcctgccagc gaagcccacc acgacgccag cgccgcgacc accaacaccg
     1141 gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg
     1201 gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc
     1261 ttggccggga cttgtggagt ccttctcctg tcactggtta tcacccttta ctgcaagcgg
     1321 cttaagcgaa gaaggatccc cgccgccgcc gccgccgccg ccgccgtcag accggtcgcc
     1381 accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
     1441 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     1501 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     1561 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaacgc ctacgtgaag
     1621 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     1681 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     1741 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     1801 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     1861 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     1921 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     1981 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     2041 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacac ttacccctac
     2101 gacgtgcccg actacgcagg ctccgcacga gtggccatcc tctggcatga gatgtggcat
     2161 gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt
     2221 gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct gaaggaaaca
     2281 tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac
     2341 atgaaatcag ggaatgtcaa ggacctcacc caagcctggg acctctatta tcatgtgttc
     2401 cgacgaatct caaagcaggc cggctgataa tctagatcat aatcagccat accacatttg
     2461 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     2521 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     2581 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2641 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     2701 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2761 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2821 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2881 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2941 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3001 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3061 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3121 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3181 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3241 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     3301 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3361 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3421 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3481 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3541 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3601 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     3661 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     3721 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     3781 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     3841 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     3901 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     3961 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4021 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4081 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4141 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     4201 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4261 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4321 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4381 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4441 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     4501 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4561 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     4621 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     4681 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     4741 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     4801 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     4861 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     4921 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4981 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5041 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5101 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5161 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5221 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5281 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5341 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5401 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5461 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5521 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5581 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5641 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5701 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagt
//
