LOCUS       sequence-339432-        5998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of plasma membrane protein Stargazin tagged with dark 
            (non-fluorescent) mCherry-FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5998)
  AUTHORS   Ferrandiz N, Downie L, Starling GP, Royle SJ
  TITLE     Endomembranes promote chromosome missegregation by ensheathing 
            misaligned chromosomes.
  JOURNAL   J Cell Biol. 2022 Jun 6;221(6). pii: 213189. doi: 
            10.1083/jcb.202203021. Epub 2022 Apr 29.
  PUBMED    35486148
REFERENCE   2  (bases 1 to 5998)
  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."; date: "2022-06-6"; volume: "221(6). pii: 213189. doi"; 
            pages: " 10.1083/jcb.202203021. Epub 2022 Apr 29"
COMMENT     Sequence Label: Stargazin-dCherry-FRB
FEATURES             Location/Qualifiers
     source          1..5998
                     /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"
     regulatory      1623..1632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1629..2333
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSNAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(1774..1792)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2042..2061
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2337..2363
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2382..2660
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2394..2663
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    2786..2907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2823..2842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2877..2896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2914..3369)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3051..3072)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3263..3282
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3396..3500
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3502..3859
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3502..3693
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3525..3545)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3710..3845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3894..4688
                     /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             3894..4688
                     /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(3948..3967)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4558..4577
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4876..4895)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4920..4967
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5296..5884
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5785..5804
                     /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 gctagcatgg
      601 ggctgtttga tcgaggtgtt caaatgcttt taaccaccgt tggtgctttc gctgccttca
      661 gcttgatgac catcgctgtg ggaaccgact attggctgta ctccagaggg gtttgcaaga
      721 ccaaaagtgt cagtgagaat gaaaccagca agaagaacga ggaagttatg acccattccg
      781 gattatggag aacctgctgc ctcgaaggga acttcaaagg tctgtgcaag caaatcgacc
      841 actttccgga agacgcggac tacgaagctg acaccgcaga gtatttcctc cgggccgtga
      901 gggcctcgag tatcttcccg atcctgagtg tgatcctgct tttcatgggt ggcctctgca
      961 tcgcagccag cgagttctac aaaccggtga actgcaggaa gagctcgaag aattcgaaaa
     1021 gcttgacacg ccacaacatc atcctgagtg ctggcatctt cttcgtgtct gcaggtctta
     1081 gtaatatcat cgggatcatc gtgtatatat cagccaatgc tggagacccc tccaagagtg
     1141 actccaaaaa gaacagctac tcctacggct ggtccttcta cttcggggcc ctgtccttca
     1201 tcatcgccga gatggtcggg gtgctggccg tgcacatgtt tatcgaccgc cacaaacagc
     1261 tgcgggccac ggcccgcgcc accgactacc tccaggcctc cgccatcacc cgcatcccca
     1321 gctaccgcta ccgctaccag cgccgcagcc gctccagctc gcgctccacc gagccctctc
     1381 actccagaga cgcctcgccc gtgggcgtga agggcttcaa caccctgccg tccacggaga
     1441 tctccatgta caccctcagt agggaccccc tgaaggctgc caccacgccc accgccacct
     1501 acaactcgga cagggataac agctttctcc aggtccacaa ctgtatccag aaggacagca
     1561 aggactctct ccacgccaac acagccaacc gccggaccac gcccgtaaag gatccaccgg
     1621 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1681 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1741 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1801 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aacgcctacg
     1861 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1921 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1981 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2041 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     2101 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2161 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     2221 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     2281 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacacttacc
     2341 cctacgacgt gcccgactac gcaggctccg cacgagtggc catcctctgg catgagatgt
     2401 ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca
     2461 tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg
     2521 aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga
     2581 agtacatgaa atcagggaat gtcaaggacc tcacccaagc ctgggacctc tattatcatg
     2641 tgttccgacg aatctcaaag caggccggct gataatctag atcataatca gccataccac
     2701 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2761 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2821 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2881 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     2941 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3001 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     3061 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     3121 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3181 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3241 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3301 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3361 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3421 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3481 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3541 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3601 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3661 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3721 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3781 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     3841 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     3901 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3961 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4021 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     4081 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4141 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4201 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4261 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4321 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4381 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4441 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4501 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4561 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4621 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4681 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4741 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4801 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4861 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     4921 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     4981 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     5041 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     5101 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     5161 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5221 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5281 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5341 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5401 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5461 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5521 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5581 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5641 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5701 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5761 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5821 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5881 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5941 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
