LOCUS       sequence-194015-        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        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1381..2079
                     /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    2670..2791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2707..2726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2761..2780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2798..3253)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2935..2956)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3147..3166
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3280..3384
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3386..3743
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3386..3577
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3409..3429)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3594..3729
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3778..4572
                     /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             3778..4572
                     /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(3832..3851)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4442..4461
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4760..4779)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4804..4851
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5180..5768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5669..5688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
      601 cagatctcga gctcaagctt cgaattcggc ggctcccgcg cgcctcccct cgcgcccgag
      661 cttcgagcca agcagcgtcc tggggagcgc gtcatggcct taccagtgac cgccttgctc
      721 ctgccgctgg ccttgctgct ccacgccgcc aggccgagcc agttccgggt gtcgccgctg
      781 gatcggacct ggaacctggg cgagacagtg gagctgaagt gccaggtgct gctgtccaac
      841 ccgacgtcgg gctgctcgtg gctcttccag ccgcgcggcg ccgccgccag tcccaccttc
      901 ctcctatacc tctcccaaaa caagcccaag gcggccgagg ggctggacac ccagcggttc
      961 tcgggcaaga ggttggggga caccttcgtc ctcaccctga gcgacttccg ccgagagaac
     1021 gagggctact atttctgctc ggccctgagc aactccatca tgtacttcag ccacttcgtg
     1081 ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc cgcgaccacc aacaccggcg
     1141 cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg
     1201 ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg
     1261 gccgggactt gtggagtcct tctcctgtca ctggttatca ccctttactg caagcggctt
     1321 aagcgaagaa ggatccccgc cgccgccgcc gccgccgccg ccgtcagacc ggtggccacc
     1381 atgagcgagc tgatcaccga gaacatgcac atgaagctgt acatggaggg caccgtgaac
     1441 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     1501 cagagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
     1561 agcttcatgt acggcagtca caccttcatc aaccacaccc agggcatccc cgacttctgg
     1621 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     1681 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     1741 atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
     1801 gaggcccaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag aaccgcgctg
     1861 gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacagatcc
     1921 aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacta cagactggaa
     1981 agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga
     2041 tactgcgacc tccctagcaa actggggcac aagcttaatc ggtacaagcg gtacaagtcc
     2101 ggactcagat ctttggctgc tgcacttgaa cgtattgaga agaactttgt cattactgac
     2161 ccaagattgc cagataatcc cattatattc gcgtccgata gtttcttgca gttgacagaa
     2221 tatagccgtg aagaaatttt gggaagaaac tgcaggtttc tacaaggtcc tgaaactgat
     2281 cgcgcgacag tgagaaaaat tagagatgcc atagataacc aaacagaggt cactgttcag
     2341 ctgattaatt atacaaagag tggtaaaaag ttctggaacc tctttcactt gcagcctatg
     2401 cgagatcaga agggagatgt ccagtacttt attggggttc agttggatgg aactgagcat
     2461 gtccgagatg ctgccgagag agaggcagtc atgctggcga agaaaactgc agaagagatt
     2521 gataaagcgg tggatacctg ggtgtagtgc ggccgcgact ctagatcata atcagccata
     2581 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2641 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2701 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2761 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     2821 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2881 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2941 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3001 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3061 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3121 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3181 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3241 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3301 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3361 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     3421 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3481 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3541 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3601 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3661 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3721 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     3781 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3841 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3901 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     3961 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4021 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4081 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4141 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4201 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4261 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     4321 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4381 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4441 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4501 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4561 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4621 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4681 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4741 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     4801 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     4861 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     4921 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     4981 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     5041 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5101 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5161 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5221 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5281 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5341 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5401 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5461 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5521 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5581 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5641 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5701 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5761 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5821 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     5881 attagttat
//
