LOCUS       sequence-423506-        7169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of ORP5(Delta-PH) fused to mScarlet-I-tagged E.
            coli DHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7169)
  AUTHORS   Ajiki M, Yoshikawa M, Miyazaki T, Kawasaki A, Aoki K, Nakatsu F, 
            Tsukiji S
  TITLE     ORP9-PH domain-based fluorescent reporters for visualizing 
            phosphatidylinositol 4-phosphate dynamics in living cells.
  JOURNAL   RSC Chem Biol. 2024 Apr 24;5(6):544-555. doi: 10.1039/d3cb00232b. 
            eCollection 2024 Jun 5.
  PUBMED    38846081
REFERENCE   2  (bases 1 to 7169)
  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"; doi: 
            "10.1039/d3cb00232b"; journalName: "RSC Chem Biol"; date: 
            "2024-04-24- 24"; volume: "5"; issue: "6"; pages: "544-555"
COMMENT     Sequence Label: pCMV-mScarlet-I-eDHFR-ORP5(Delta-PH)
FEATURES             Location/Qualifiers
     source          1..7169
                     /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..1308
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(746..764)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1014..1033
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3957..4078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3994..4013)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4048..4067
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4085..4540)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4222..4243)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4434..4453
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4567..4671
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4673..5030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4673..4864
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4696..4716)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4881..5016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5065..5859
                     /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             5065..5859
                     /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(5119..5138)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5729..5748
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6047..6066)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6091..6138
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6467..7055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6956..6975
                     /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 gcagtgatca aggagttcat gcggttcaag
      661 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
      721 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      781 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt caccaagcac
      841 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc
      901 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
      961 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     1021 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc
     1081 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg
     1141 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc
     1201 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     1261 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc cggactcaga
     1321 tctcgaagcg cggccgcggg agcaggagga gcagctcgag cggcgatgat ttctctgatt
     1381 gccgctctgg ccgtggacag agtgatcggc atggaaaacg ccatgccttg gaacctgcct
     1441 gccgatctgg cctggttcaa gcggaatacc ctgaacaagc ccgtgatcat gggcagacac
     1501 acctgggagt ctatcggcag acctctgcct ggccggaaga acatcattct gtctagccag
     1561 cctggcaccg acgatagagt gacatgggtc aagagcgtgg acgaggccat tgctgcttgc
     1621 ggagatgtgc ctgagatcat ggttatcggc ggaggccggg tgtacgagca gtttctgcct
     1681 aaggctcaga agctgtacct gacacacatc gacgccgagg tggaaggcga tacacacttc
     1741 cccgactacg agcccgatga ctgggagagc gtgttcagcg agtttcacga cgccgacgct
     1801 cagaacagcc acagctactg cttcgagatc ctggaaagac gcgccggatc tgggggcggt
     1861 accggagctg gagggagcgg cgggggaagt cgagctcaag cttcgtgcaa gccgggccga
     1921 gacggggagc cagggacctc gccagacgca tcaccctcat cgctctgtgg gctgccagcc
     1981 tcagccaccg tccacccaga ccaagacctg ttcccactga acgggtcttc cctggagaac
     2041 gatgcattct cagacaagtc ggagagagag aaccctgagg agtcagatac cgagacccag
     2101 gaccatagcc ggaagacgga gagtggcagc gaccagtcag agacccctgg ggccccggtg
     2161 cggagaggga ccacctatgt ggagcaggtc caggaggagc tgggggagct gggcgaggcg
     2221 tcccaggtgg agacagtgtc agaggagaac aagagtctga tgtggaccct gctgaagcag
     2281 ctacggccag gcatggacct gtcccgcgtg gtgctaccca cgttcgtact ggagccgcgc
     2341 tccttcctga acaagctctc cgactactac taccacgcag acctgctctc cagggctgcg
     2401 gtggaggagg atgcctacag ccgcatgaag ctggtgctgc ggtggtacct gtctggcttc
     2461 tacaagaagc ccaagggaat caagaagccg tacaacccca tcctggggga gaccttccgc
     2521 tgctgctggt tccacccgca gactgacagc cgcacattct acatagcaga gcaggtgtcc
     2581 caccacccgc ccgtgtctgc cttccacgtc agcaaccgga aggacggctt ctgcatcagt
     2641 ggcagcatca cagccaagtc caggttttat gggaactcgc tgtcggcgct gctggacggc
     2701 aaagccacgc tcaccttcct gaaccgagcc gaggattaca cccttaccat gccctacgcc
     2761 cactgcaaag gaatcctgta tggcacgatg accctggagc tgggtgggaa ggtcaccatc
     2821 gagtgtgcga agaacaactt ccaggcccag ctggaattca aactcaagcc cttcttcggg
     2881 ggtagcacca gcatcaacca gatctcggga aagatcacgt cgggagagga agtcctggcg
     2941 agcctcagtg gccactggga cagggacgtg tttatcaagg aggaagggag cggaagcagt
     3001 gcgcttttct ggaccccgag cggggaggtc cgcagacaga ggctgaggca gcacacggtg
     3061 ccgctggagg agcagacgga gctggagtcc gagaggctct ggcagcacgt caccagggcc
     3121 atcagcaagg gcgaccagca cagggccaca caggagaagt ttgcactgga ggaggcacag
     3181 cggcagcggg cccgtgagcg gcaggagagc ctcatgccct ggaagccgca gctgttccac
     3241 ctggacccca tcacccagga gtggcactac cgatacgagg accacagccc ctgggacccc
     3301 ctgaaggaca tcgcccagtt tgagcaagac gggatcctgc ggaccttgca gcaggaggcc
     3361 gtggcccgcc agaccacctt cctgggcagc ccagggccca ggcacgagag gtctggccca
     3421 gaccagcggc ttcgcaaggc cagcgaccag ccctccggcc acagccaggc cacggagagc
     3481 agcggatcca cgcctgagtc ctgcccagag ctctcagacg aggagcagga tggtgacttt
     3541 gtccctggcg gtgagagccc atgccctcgg tgcaggaagg aggcgcggcg gctgcaggcc
     3601 ctgcacgagg ccatcctctc catccgagag gcccagcagg agctgcacag gcacctctcg
     3661 gccatgctga gctccacggc acgggcagca caggcaccga ccccaggcct cctgcagagc
     3721 ccccgatcct ggttcctgct ctgcgtgttc ctggcgtgtc agctgttcat taaccacatc
     3781 ctcaaataga agcttcgaat tctgcagtcg acggtaccgc gggcccggga tccaccggat
     3841 ctagataact gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     3901 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     3961 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4021 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     4081 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4141 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     4201 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4261 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4321 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     4381 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     4441 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     4501 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     4561 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4621 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     4681 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     4741 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     4801 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     4861 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4921 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4981 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     5041 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     5101 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     5161 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     5221 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     5281 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     5341 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     5401 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     5461 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     5521 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     5581 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5641 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5701 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5761 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5821 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5881 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5941 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     6001 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     6061 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     6121 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     6181 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     6241 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     6301 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6361 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6421 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6481 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6541 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6601 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6661 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6721 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6781 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6841 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6901 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6961 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7021 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7081 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7141 ctgtggataa ccgtattacc gccatgcat
//
