LOCUS       sequence-194001-        5283 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 5283)
  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 5283)
  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: Myc-CD4-mCherry-HA-FRB
FEATURES             Location/Qualifiers
     source          1..5283
                     /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             685..714
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      892..901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             898..1602
                     /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(1043..1061)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1311..1330
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1606..1632
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1651..1929
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1663..1932
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    2069..2190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2106..2125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2160..2179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2197..2652)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2334..2355)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2546..2565
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2679..2783
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2785..3142
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2785..2976
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2808..2828)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2993..3128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3177..3971
                     /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             3177..3971
                     /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(3231..3250)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3841..3860
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4159..4178)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4203..4250
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4579..5167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5068..5087
                     /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 tagcaaggcc
      601 acaatgaacc ggggagtccc ttttaggcac ttgcttctgg tgctgcaact ggcgctcctc
      661 ccagcagcca ctcagggaaa gaaagaacaa aaactcatct cagaagagga tctgtgtctg
      721 ctgagtgact cgggacaggt cctgctggaa tccaacatca aggttctgcc cacatggtcc
      781 accccggtgc agccaatggc cctgattgtg ctggggggcg tcgccggcct cctgcttttc
      841 attgggctag gcatcttctt ctgtgtcagg tgccggcacc gaaggcgcca agccaccatg
      901 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
      961 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1021 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1081 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1141 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1201 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1261 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1321 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1381 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1441 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1501 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     1561 gccgagggcc gccactccac cggcggcatg gacgagctgt acacttaccc ctacgacgtg
     1621 cccgactacg caggctccgc acgagtggcc atcctctggc atgagatgtg gcatgaaggc
     1681 ctggaagagg catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg
     1741 ctggagccct tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt
     1801 aatcaggcct atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa
     1861 tcagggaatg tcaaggacct cacccaagcc tgggacctct attatcatgt gttccgacga
     1921 atctcaaagc aggaccggtg gatccaccgg atctagataa ctgatcataa tcagccatac
     1981 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2041 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2101 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2161 tggtttgtcc aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt
     2221 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2281 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2341 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2401 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2461 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2521 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2581 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2641 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2701 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2761 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     2821 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2881 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2941 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3001 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3061 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3121 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3181 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3241 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3301 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3361 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3421 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3481 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3541 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3601 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3661 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3721 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3781 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3841 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3901 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3961 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4021 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4081 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4141 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4201 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4261 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     4321 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4381 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4441 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4501 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4681 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5221 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     5281 tta
//
