LOCUS       sequence-368434-        5296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FRB-mCherry-tagged ER protein Sec61beta in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5296)
  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 5296)
  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-Sec61-beta
FEATURES             Location/Qualifiers
     source          1..5296
                     /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"
     CDS             1633..1923
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     polyA_signal    2084..2205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2121..2140)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2175..2194
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2212..2667)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2349..2370)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2561..2580
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2694..2798
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2800..3157
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2800..2991
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2823..2843)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3008..3143
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3192..3986
                     /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             3192..3986
                     /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(3246..3265)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3856..3875
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4174..4193)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4218..4265
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4594..5182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5083..5102
                     /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 ctatgcctgg tccgaccccc agtggcacta acgtgggatc ctcagggcgc
     1681 tctcccagca aagcagtggc cgcccgggcg gcgggatcca ctgtccggca gaggaaaaat
     1741 gccagctgtg ggacaaggag tgcaggccgc acaacctcgg caggcaccgg ggggatgtgg
     1801 cgattctaca cagaagattc acctgggctc aaagttggcc ctgttccagt attggttatg
     1861 agtcttctgt tcatcgcttc tgtatttatg ttgcacattt ggggcaagta cactcgttcg
     1921 taggaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat
     1981 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2041 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     2101 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2161 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     2221 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2281 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2341 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2401 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2461 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2521 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2581 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2641 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     2701 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2761 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     2821 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2881 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2941 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3001 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3061 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3121 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     3181 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3241 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3301 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3361 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3421 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3481 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3541 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3601 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3661 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     3721 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3781 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3841 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3901 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3961 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4021 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4081 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4141 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     4201 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     4261 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     4321 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     4381 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     4441 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4501 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4561 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4621 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4681 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4741 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4801 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4861 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4921 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4981 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5041 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5101 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5161 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5221 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5281 tattaccgcc atgcat
//
