LOCUS       sequence-423510-        7938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of green fluorescent PI4P reporter 
            AcGFP1-ORP9-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  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 7938)
  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: pPBbsr-AcGFP1-ORP9-PH
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1667
                     /label=chicken beta-actin promoter
     intron          1668..2684
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2608..2630
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2692..2711
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2768..2777
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2774..3490
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    4002..4551
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4146..4163)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4373..4392
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(5022..5039)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5028..5252
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5299..5315)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5323..5339
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5347..5353,5354..5371,5372..5377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5392..5413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5647..5709
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5750..5770)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5750..5768)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5789..5805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5813..5829
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5837..5843,5844..5861,5862..5867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5882..5903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6020..6037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6191..6779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6271..6290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6950..7741,7742..7810))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7573..7592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7811..7915)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1501 gggggggggg gsgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
     1561 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     1621 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1681 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1741 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1801 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     1861 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     1921 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     1981 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     2041 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     2101 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     2161 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     2221 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     2281 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     2341 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     2401 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     2461 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     2521 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     2581 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2641 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2701 tggttattgt gctgtctcat cattttggca aagaattggg atcgaattgt agctagcgct
     2761 accggtcgcc accatggtga gcaagggcgc cgagctgttc accggcatcg tgcccatcct
     2821 gatcgagctg aatggcgatg tgaatggcca caagttcagc gtgagcggcg agggcgaggg
     2881 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcctgt
     2941 gccctggccc accctggtga ccaccctgag ctacggcgtg cagtgcttct cacgctaccc
     3001 cgatcacatg aagcagcacg acttcttcaa gagcgccatg cctgagggct acatccagga
     3061 gcgcaccatc ttcttcgagg atgacggcaa ctacaagtcg cgcgccgagg tgaagttcga
     3121 gggcgatacc ctggtgaatc gcatcgagct gaccggcacc gatttcaagg aggatggcaa
     3181 catcctgggc aataagatgg agtacaacta caacgcccac aatgtgtaca tcatgaccga
     3241 caaggccaag aatggcatca aggtgaactt caagatccgc cacaacatcg aggatggcag
     3301 cgtgcagctg gccgaccact accagcagaa tacccccatc ggcgatggcc ctgtgctgct
     3361 gcccgataac cactacctgt ccacccagag cgccctgtcc aaggacccca acgagaagcg
     3421 cgatcacatg atctacttcg gcttcgtgac cgccgccgcc atcacccacg gcatggatga
     3481 gctgtacaag tccggactca gatctggtgg cggaggctcg ggcggaggtg ggtcgggtgg
     3541 cggcggatct cgagctcaag cttcgatggc gtccatcgtg gaagggccgc tgagcaaatg
     3601 gactaacgtg atgaagggat ggcagtatcg ttggttcgtg ctggactaca atgcagggct
     3661 gctctcctac tacacgtcca aggacaaaat gatgagaggc tctcgaagag gatgcgttag
     3721 actcagagga gctgtgattg gtatagacga cgaggacgac agcaccttca caatcactgt
     3781 cgatcagaaa accttccact tccaggctcg agatgcagac gagcgagaga agtggatcca
     3841 tgccttagaa gaaactattc ttcgccatac tcttcagctt caaggtttgg attcaggatg
     3901 acccgggatc ctgcagtcga cgggccgcgg taacaattgt taactaactt aagctagcaa
     3961 cggtttccct ctagcgggat caattccgcc ccccccccct aacgttactg gccgaagccg
     4021 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     4081 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     4141 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     4201 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     4261 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     4321 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     4381 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     4441 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     4501 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgataat accatggtca
     4561 tgaaaacatt taacatttct caacaagatc tagaattagt agaagtagcg acagagaaga
     4621 ttacaatgct ttatgaggat aataaacatc atgtgggagc ggcaattcgt acgaaaacag
     4681 gagaaatcat ttcggcagta catattgaag cgtatatagg acgagtaact gtttgtgcag
     4741 aagccattgc gattggtagt gcagtttcga atggacaaaa ggattttgac acgattgtag
     4801 ctgttagaca cccttattct gacgaagtag atagaagtat tcgagtggta agtccttgtg
     4861 gtatgtgtag ggagttgatt tcagactatg caccagattg ttttgtgtta atagaaatga
     4921 atggcaagtt agtcaaaact acgattgaag aactcattcc actcaaatat acccgaaatt
     4981 aaaagtcgag atgcatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag
     5041 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     5101 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     5161 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5221 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc
     5281 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5341 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5401 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5461 ccagcggatc ttaaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt
     5521 taataaataa ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta
     5581 aatataataa aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat
     5641 aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc
     5701 tttctagggt taatctagta tacgcgttta attaatccag cttttgttcc ctttagtgag
     5761 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5821 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5881 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5941 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6001 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6061 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6121 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6181 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6241 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6301 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6361 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6421 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6481 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6541 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6601 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6661 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6721 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6781 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6841 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6901 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6961 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7021 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7081 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7141 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7201 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7261 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7321 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7381 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7441 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7501 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7561 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7621 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7681 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7741 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7801 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7861 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7921 ttccccgaaa agtgccac
//
