LOCUS       sequence-380169-        5370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry tagged with two FRB mutants (T2098L) in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5370)
  AUTHORS   Obashi K, Sochacki KA, Strub MP, Taraska JW
  TITLE     A conformational switch in clathrin light chain regulates lattice 
            structure and endocytosis at the plasma membrane of mammalian cells.
  JOURNAL   Nat Commun. 2023 Feb 9;14(1):732. doi: 10.1038/s41467-023-36304-7.
  PUBMED    36759616
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: "Nat 
            Commun."; date: "2023-02-9"; volume: "14(1):732. doi"; pages: " 
            10.1038/s41467-023-36304-7"
COMMENT     Sequence Label: mCherry-FRBx2
FEATURES             Location/Qualifiers
     source          1..5370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      644..653
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             650..1357
                     /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(795..813)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1063..1082
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1370..1648
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1664..1942
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     misc_feature    1989..2041
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2165..2286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2202..2221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2256..2275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2293..2748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2430..2451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2642..2661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2775..2879
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2881..3238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2881..3072
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2904..2924)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3089..3224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3273..4067
                     /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             3273..4067
                     /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(3327..3346)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3937..3956
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4255..4274)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4299..4346
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4675..5263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5164..5183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc tcgagctcaa gcttcgaatt ccggccacca tggtgagcaa
      661 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
      721 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      781 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
      841 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
      901 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      961 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     1021 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     1081 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     1141 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     1201 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     1261 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     1321 ccgccactcc accggcggca tggacgagct gtacaaggga gcaagtggaa tcctctggca
     1381 tgagatgtgg catgaaggcc tggaagaggc atctcgtttg tactttgggg aaaggaacgt
     1441 gaaaggcatg tttgaggtgc tggagccctt gcatgctatg atggaacggg gcccccagac
     1501 tctgaaggaa acatccttta atcaggccta tggtcgagat ttaatggagg cccaagagtg
     1561 gtgcaggaag tacatgaaat cagggaatgt caaggacctc ctccaagcct gggacctcta
     1621 ttatcatgtg ttccgacgaa tctcaaagaa gggagcaagt ggaatcctct ggcatgagat
     1681 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     1741 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     1801 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag
     1861 gaagtacatg aaatcaggga atgtaaagga cctcctccaa gcctgggacc tctattatca
     1921 tgtgttccga cgaatctcaa agagtgctgg tggtagtgct ggtggtagtg ctggtggtac
     1981 tggtggtcct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc
     2041 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     2101 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     2161 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2221 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2281 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attagcgtta aatttttgtt
     2341 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2401 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2461 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2521 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2581 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2641 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2701 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2761 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2881 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2941 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3001 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3061 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3121 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3181 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3241 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3301 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3361 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3421 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3481 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3541 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3601 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3661 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3721 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3781 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3841 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3901 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3961 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4021 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4081 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4141 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4201 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4261 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4321 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4381 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4441 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4501 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4561 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4621 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4681 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4741 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4801 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4861 ctctgtatca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4921 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4981 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5041 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5101 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5161 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5221 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5281 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5341 ccctgattct gtggataacc gtattaccgc
//
