LOCUS       sequence-424113-        6691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of near-IR fluorescent PI4P reporter 
            iRFP713-ORP9-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6691)
  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 6691)
  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-iRFP713-ORP9-PH
FEATURES             Location/Qualifiers
     source          1..6691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..309
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     464..484
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      552..561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             558..1505
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    2059..2180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2096..2115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2150..2169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2187..2642)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2324..2345)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2536..2555
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2669..2773
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2775..3132
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2775..2966
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2798..2818)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2983..3118
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3167..3961
                     /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             3167..3961
                     /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(3221..3240)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3831..3850
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4149..4168)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4193..4240
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4569..5157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5176..5192
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5176..5192
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5515..5532)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5515..5531)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5786..5805
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5786..5804
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5893..5911)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     rep_origin      5934..6522
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
       61 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      121 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      181 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
      541 cgctaccggt cgccaccatg gcggaaggat ctgtcgccag gcagcctgac ctcttgacct
      601 gcgacgatga gccgatccat atccccggtg ccatccaacc gcatggactg ctgctcgccc
      661 tcgccgccga catgacgatc gttgccggca gcgacaacct tcccgaactc accggactgg
      721 cgatcggcgc cctgatcggc cgctctgcgg ccgatgtctt cgactcggag acgcacaacc
      781 gtctgacgat cgccttggcc gagcccgggg cggccgtcgg agcaccgatc actgtcggct
      841 tcacgatgcg aaaggacgca ggcttcatcg gctcctggca tcgccatgat cagctcatct
      901 tcctcgagct cgagcctccc cagcgggacg tcgccgagcc gcaggcgttc ttccgccgca
      961 ccaacagcgc catccgccgc ctgcaggccg ccgaaacctt ggaaagcgcc tgcgccgccg
     1021 cggcgcaaga ggtgcggaag attaccggct tcgatcgggt gatgatctat cgcttcgcct
     1081 ccgacttcag cggcgaagtg atcgcagagg atcggtgcgc cgaggtcgag tcaaaactag
     1141 gcctgcacta tcctgcctca accgtgccgg cgcaggcccg tcggctctat accatcaacc
     1201 cggtacggat cattcccgat atcaattatc ggccggtgcc ggtcacccca gacctcaatc
     1261 cggtcaccgg gcggccgatt gatcttagct tcgccatcct gcgcagcgtc tcgcccgtcc
     1321 atctggagtt catgcgcaac ataggcatgc acggcacgat gtcgatctcg attttgcgcg
     1381 gcgagcgact gtggggattg atcgtttgcc atcaccgaac gccgtactac gtcgatctcg
     1441 atggccgcca agcctgcgag ctagtcgccc aggttctggc ctggcagatc ggcgtgatgg
     1501 aagagctgta caagtccgga ctcagatctg gtggcggagg ctcgggcgga ggtgggtcgg
     1561 gtggcggcgg atctcgagct caagcttcga tggcgtccat cgtggaaggg ccgctgagca
     1621 aatggactaa cgtgatgaag ggatggcagt atcgttggtt cgtgctggac tacaatgcag
     1681 ggctgctctc ctactacacg tccaaggaca aaatgatgag aggctctcga agaggatgcg
     1741 ttagactcag aggagctgtg attggtatag acgacgagga cgacagcacc ttcacaatca
     1801 ctgtcgatca gaaaaccttc cacttccagg ctcgagatgc agacgagcga gagaagtgga
     1861 tccatgcctt agaagaaact attcttcgcc atactcttca gcttcaaggt ttggattcag
     1921 gatgacccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     1981 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     2041 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2101 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2161 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     2221 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2281 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2341 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2401 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2461 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2521 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2581 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2641 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2701 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2761 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     2821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2881 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2941 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3001 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3061 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3121 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3181 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3241 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3301 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     3361 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3421 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3481 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3541 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3601 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3661 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     3721 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3781 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3841 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3901 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3961 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4021 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4081 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     4141 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     4201 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     4261 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     4321 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     4381 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     4441 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4501 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4561 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4621 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4681 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4741 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4801 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4861 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4921 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4981 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5041 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5101 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5161 gccagcaacg cggcccagga aacagctatg accatgcccg ggtaagtgac tagcggaaac
     5221 acagaaaaaa gcccgcacct gacagtgcgg gctttttttt tcgaccaaag gctccgaaga
     5281 gccgagtgtc ttcggttcga gaccgaattc gcgagctctc gctagccagt ttaaacaatt
     5341 gcggccgcgt actagtctat cgatttggat ccaccctgca ggaagtctag accgtcgaca
     5401 cggtctctga gagaagacac gtcgctcttc gacgtaagta actgactcgg taccaaattc
     5461 cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtcctaag cttaactggc
     5521 cgtcgtttta caacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
     5581 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
     5641 tcactggata taccaccgtc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     5701 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     5761 cgatgagtgg cagggcgggg cgtaataata cgactcacta tagggtaact aattgaggcg
     5821 cgcctaccac cgtcaaaaaa aacggcgctt tttagcgccg tttttatttt tcaaccttcc
     5881 taggtaagtg accctttagt gagggttaat ttagaaaaga tcaaaggatc ttcttgagat
     5941 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6001 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6061 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6121 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6181 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6241 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6301 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6361 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6421 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6481 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6541 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6601 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac
     6661 ggggtcatta gttcatagcc catatatgga g
//
