LOCUS       sequence-368440-        5370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FRB-mCherry on Golgi membrane with Giantin 
            transmembrane (residues 3131-3259).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5370)
  AUTHORS   Kuey C, Sittewelle M, Larocque G, Hernandez-Gonzalez M, Royle SJ
  TITLE     Recruitment of clathrin to intracellular membranes is sufficient for
            vesicle formation.
  JOURNAL   Elife. 2022 Jul 19;11. pii: 78929. doi: 10.7554/eLife.78929.
  PUBMED    35852853
REFERENCE   2  (bases 1 to 5370)
  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: "Elife."; 
            date: "2022-07-19"; volume: "11. pii: 78929. doi"; pages: " 
            10.7554/eLife.78929"
COMMENT     Sequence Label: FRB-mCherry-Giantin
FEATURES             Location/Qualifiers
     source          1..5370
                     /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             607..885
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             619..888
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..1617
                     /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
                     EGRHSTGGMDELYK"
     primer_bind     complement(1055..1073)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1323..1342
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2158..2279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2195..2214)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2249..2268
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2286..2741)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2423..2444)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2635..2654
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2768..2872
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2874..3231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2874..3065
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2897..2917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3082..3217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3266..4060
                     /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             3266..4060
                     /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(3320..3339)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3930..3949
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4248..4267)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4292..4339
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4668..5256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5157..5176
                     /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 gctagcggcc
      601 accatgatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
      661 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
      721 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
      781 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcacc
      841 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagcaggc cggcccaccg
      901 gtcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      961 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     1021 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     1081 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     1141 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     1201 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1261 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1321 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1381 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1441 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1501 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1561 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtcc
     1621 ggactcagat ctcgaggaga accgcagcaa agcttttctg aagctcagca gcagctatgc
     1681 aacaccagac aggaagtgaa tgaattaagg aagctgctgg aagaagaacg agaccaaaga
     1741 gtggctgctg agaatgctct ctctgtggcc gaggagcaga tcagacggtt agagcacagt
     1801 gaatgggact cttcccggac tcctatcatt ggctcctgtg gcactcagga gcaggcactg
     1861 ttaatagatc ttacaagcaa cagttgtcga aggacccgga gtggcgttgg atggaagcga
     1921 gtcctgcgtt cactctgtca ttcacggacc cgagtgccac ttctagcagc catctacttt
     1981 ctaatgattc atgtcctgct cattctgtgt tttacgggcc atctataggg atccaccgga
     2041 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2101 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2161 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2221 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2281 cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2341 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2401 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2461 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2521 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2581 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2641 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2701 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     2761 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2821 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     2881 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2941 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3001 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3061 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3121 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3181 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3241 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3301 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3361 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3421 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3481 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3541 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3601 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3661 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3721 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3781 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3841 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3901 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3961 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4021 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4081 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4141 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4201 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4261 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4321 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4381 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4441 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4501 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4561 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4621 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4681 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4741 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4801 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4861 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4921 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4981 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5041 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5101 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5161 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5221 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5281 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5341 tctgtggata accgtattac cgccatgcat
//
