LOCUS       sequence-189384-        5889 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 5889)
  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 5889)
  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-pTagRFP657-LOVpep (T406A, T407A, I532A)
FEATURES             Location/Qualifiers
     source          1..5889
                     /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"
     CDS             1388..2086
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    2677..2798
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2714..2733)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2768..2787
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2805..3260)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2942..2963)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3154..3173
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3287..3391
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3393..3750
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3393..3584
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3416..3436)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3601..3736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3785..4579
                     /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             3785..4579
                     /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(3839..3858)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4449..4468
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4767..4786)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4811..4858
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5187..5775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5676..5695
                     /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 ggccaccatg agcgagctga tcaccgagaa catgcacatg aagctgtaca tggagggcac
     1441 cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     1501 ccagacccag agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     1561 ggctaccagc ttcatgtacg gcagtcacac cttcatcaac cacacccagg gcatccccga
     1621 cttctggaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     1681 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     1741 cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
     1801 cggctgggag gcccacaccg agatgctgta ccccgctgac ggcggcctgg aaggcagaac
     1861 cgcgctggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttca agaccacata
     1921 cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg tggactacag
     1981 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt
     2041 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaatcggt acaagcggta
     2101 caagtccgga ctcagatctt tggctgctgc acttgaacgt attgagaaga actttgtcat
     2161 tactgaccca agattgccag ataatcccat tatattcgcg tccgatagtt tcttgcagtt
     2221 gacagaatat agccgtgaag aaattttggg aagaaactgc aggtttctac aaggtcctga
     2281 aactgatcgc gcgacagtga gaaaaattag agatgccata gataaccaaa cagaggtcac
     2341 tgttcagctg attaattata caaagagtgg taaaaagttc tggaacctct ttcacttgca
     2401 gcctatgcga gatcagaagg gagatgtcca gtactttatt ggggttcagt tggatggaac
     2461 tgagcatgtc cgagatgctg ccgagagaga ggcagtcatg ctggcgaaga aaactgcaga
     2521 agagattgat aaagcggtgg atacctgggt gtagtgcggc cgcgactcta gatcataatc
     2581 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     2641 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2701 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2761 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     2821 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2881 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2941 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     3001 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     3061 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     3121 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     3181 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     3241 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3361 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     3421 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3481 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3541 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3601 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3661 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3721 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     3781 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3841 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3901 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3961 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4021 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4081 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4141 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4201 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4261 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     4321 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4381 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4441 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4501 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4561 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4621 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4681 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4741 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4801 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     4861 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     4921 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     4981 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     5041 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5101 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5161 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5221 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5281 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5341 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5401 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5461 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5521 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5581 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5641 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5701 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5761 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5821 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5881 gccatgcat
//
