LOCUS       sequence-434122-        7576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses a mutant ORP5 that is unable to bind to PI4P with an 
            mCherry fluorophore and an FKBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7576)
  AUTHORS   Doyle CP, Rectenwald A, Timple L, Hammond GRV
  TITLE     Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a 
            Major Player in PM PI4P Turnover.
  JOURNAL   Contact (Thousand Oaks). 2024 Feb 7;7:25152564241229272. doi: 
            10.1177/25152564241229272. eCollection 2024 Jan-Dec.
  PUBMED    38327560
REFERENCE   2  (bases 1 to 7576)
  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: "Contact 
            (Thousand Oaks)."; date: "2024-02-7"; volume: "7:25152564241229272. 
            doi"; pages: " 10.1177/25152564241229272. eCollection 2024 Jan-Dec"
COMMENT     Sequence Label: mCherry-C1-ORP5-H478-9A-deltaTMD-FKBP
FEATURES             Location/Qualifiers
     source          1..7576
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /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(758..776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1026..1045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(3907..3909,3910..4230)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     polyA_signal    4364..4485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4401..4420)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4455..4474
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4492..4947)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4629..4650)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4841..4860
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4974..5078
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5080..5437
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5080..5271
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      5288..5423
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5472..6266
                     /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             5472..6266
                     /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(5526..5545)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6136..6155
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6454..6473)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6498..6545
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6874..7462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7363..7382
                     /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 gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tccggactca gatctcgagc gaaggaggag gccttcctcc ggcgccgctt ctccctgtgt
     1381 ccaccttcct ccacccctca gaaagtcgac ccccggaagc tcacccggaa cttgctcctc
     1441 agcggagaca atgagctcta cccactcagc ccagggaagg acatggagcc caacggcccg
     1501 tcgctgccca gggatgaagg gcccccgacc ccaagctctg ccacgaaggt gccaccggca
     1561 gagtacaggc tgtgcaacgg gtcagacaag gaatgtgtgt cccccaccgc cagggtcacc
     1621 aagaaggaga ctctcaaggc gcagaaggag aactaccggc aggagaagaa gcgcgccaca
     1681 cggcagctgc tcagcgctct gacagacccc agcgtggtca tcatggctga cagcctgaag
     1741 atccgcggca ccctgaagag ctggaccaag ctgtggtgcg tgctgaagcc gggggtgctg
     1801 ctcatctaca agacgcccaa ggtgggccag tgggtgggca cggtgctgct gcactgctgc
     1861 gagctcatcg agcggccctc caagaaggac ggcttctgct tcaagctctt ccacccgctg
     1921 gatcagtccg tctgggccgt gaagggcccc aaaggtgaga gcgtgggctc catcacacag
     1981 cccctgccca gcagctacct gatcttcagg gccgcctccg agtcagatgg tcgctgctgg
     2041 ctggacgccc tggagctggc cctgcgctgc tctagcctac tgagactggg cacctgcaag
     2101 ccgggccgag acggggagcc agggacctcg ccagacgcat caccctcatc gctctgtggg
     2161 ctgccagcct cagccaccgt ccacccagac caagacctgt tcccactgaa cgggtcttcc
     2221 ctggagaacg atgcattctc agacaagtcg gagagagaga accctgagga gtcagatacc
     2281 gagacccagg accatagccg gaagacggag agtggcagcg accagtcaga gacccctggg
     2341 gccccggtgc ggagagggac cacctatgtg gagcaggtcc aggaggagct gggggagctg
     2401 ggcgaggcgt cccaggtgga gacagtgtca gaggagaaca agagtctgat gtggaccctg
     2461 ctgaagcagc tacggccagg catggacctg tcccgcgtgg tgctacccac gttcgtactg
     2521 gagccgcgct ccttcctgaa caagctctcc gactactact accacgcaga cctgctctcc
     2581 agggctgcgg tggaggagga tgcctacagc cgcatgaagc tggtgctgcg gtggtacctg
     2641 tctggcttct acaagaagcc caagggaatc aagaagccgt acaaccccat cctgggggag
     2701 accttccgct gctgctggtt ccacccgcag actgacagcc gcacattcta catagcagag
     2761 caggtgtccg ccgccccgcc cgtgtctgcc ttccacgtca gcaaccggaa ggacggcttc
     2821 tgcatcagtg gcagcatcac agccaagtcc aggttttatg ggaactcgct gtcggcgctg
     2881 ctggacggca aagccacgct caccttcctg aaccgagccg aggattacac ccttaccatg
     2941 ccctacgccc actgcaaagg aatcctgtat ggcacgatga ccctggagct gggtgggaag
     3001 gtcaccatcg agtgtgcgaa gaacaacttc caggcccagc tggaattcaa actcaagccc
     3061 ttcttcgggg gtagcaccag catcaaccag atctcgggaa agatcacgtc gggagaggaa
     3121 gtcctggcga gcctcagtgg ccactgggac agggacgtgt ttatcaagga ggaagggagc
     3181 ggaagcagtg cgcttttctg gaccccgagc ggggaggtcc gcagacagag gctgaggcag
     3241 cacacggtgc cgctggagga gcagacggag ctggagtccg agaggctctg gcagcacgtc
     3301 accagggcca tcagcaaggg cgaccagcac agggccacac aggagaagtt tgcactggag
     3361 gaggcacagc ggcagcgggc ccgtgagcgg caggagagcc tcatgccctg gaagccgcag
     3421 ctgttccacc tggaccccat cacccaggag tggcactacc gatacgagga ccacagcccc
     3481 tgggaccccc tgaaggacat cgcccagttt gagcaagacg ggatcctgcg gaccttgcag
     3541 caggaggccg tggcccgcca gaccaccttc ctgggcagcc cagggcccag gcacgagagg
     3601 tctggcccag accagcggct tcgcaaggcc agcgaccagc cctccggcca cagccaggcc
     3661 acggagagca gcggatccac gcctgagtcc tgcccagagc tctcagacga ggagcaggat
     3721 ggtgactttg tccctggcgg tgagagccca tgccctcggt gcaggaagga ggcgcggcgg
     3781 ctgcaggccc tgcacgaggc catcctctcc atccgagagg cccagcagga gctgcacagg
     3841 cacctctcgg ccatgctgag ctccacggca cgggcagcac aggcaccgac cccaggcctc
     3901 ctgcagatgg gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag
     3961 cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaatttgat
     4021 tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga
     4081 ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct
     4141 ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc
     4201 gtcttcgatg tggagcttct aaaactggaa cccgggatcc accggatcta gataactgat
     4261 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4321 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4381 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4441 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     4501 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4561 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4621 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4681 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4741 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4801 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4861 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4921 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4981 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5041 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     5101 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5161 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     5221 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     5281 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     5341 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     5401 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     5461 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5521 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5581 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5641 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5701 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5761 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5821 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5881 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5941 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     6001 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     6061 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     6121 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     6181 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     6241 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     6301 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6361 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6421 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     6481 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     6541 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     6601 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     6661 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     6721 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6781 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6841 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6901 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6961 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7021 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7081 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7141 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7201 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7261 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7321 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7381 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7441 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7501 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7561 tattaccgcc atgcat
//
