LOCUS       sequence-399471-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses recruitable fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  AUTHORS   Wills RC, Doyle CP, Zewe JP, Pacheco J, Hansen SD, Hammond GRV
  TITLE     A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at
            the plasma membrane.
  JOURNAL   J Cell Sci. 2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494.
  PUBMED    37534432
REFERENCE   2  (bases 1 to 6977)
  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 Sci.
            2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494."
COMMENT     Sequence Label: iRFP713-FRB-PIP5K1A
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..149
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        166..357
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     314..334
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(359..463)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      490..945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(577..596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     787..808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    952..1073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(963..982)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1017..1036
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2951..3229)
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             complement(3278..4225)
                     /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"
     regulatory      4222..4231
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4270..4473)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4299..4319)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4474..4777
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(4952..5540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5032..5051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5869..5916
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5941..5960
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(6148..6942)
                     /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             complement(6148..6942)
                     /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(6259..6278)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6869..6888
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6977,1..357))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggcc
       61 gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg
      121 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct
      181 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
      241 cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
      301 ggggactttc cacaccctaa ctgacacaca ttccacagct ggttctttcc gcctcaggac
      361 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
      421 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
      481 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
      541 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      601 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      661 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      721 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
      781 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
      841 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
      901 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttacgcc ttaagataca
      961 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     1021 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     1081 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     1141 agtaaaacct ctacaaatgt ggtatggctg attatgatca gttatctaga tccggtggat
     1201 cccgttaatg ggtgaactct gactctgcaa cttcaagctt ttccaaggtt gtacttgtag
     1261 ttagcatttg caaagtctct ccaactagag gtgagaaact ggggtcagga ataggcgagc
     1321 cctcactgat ttcctccaaa ggtggagtct gaggtaaaac atcaggacga ccaaggtgaa
     1381 cgcctggctc cacttctgcc tttgttgtca cttgtgcctt gtgttcccca gagaccgatg
     1441 gctggtaagt aatgcaggag ttgccactgg agcctgctcg ccgagagaaa gatgagccag
     1501 accgaaactt tttggaagga gaaggcttca agggaatctt cttaaatact gtgttgcaca
     1561 tgaagcgctg gaaccgttca gcgtagaagc ctgggcgatg cactgagaca gtgtctccgt
     1621 catgtaccag ggctttccaa gagtgctcca acttcttaac aaacctgtaa gactgtagaa
     1681 tgtcaatgat gccaatataa agcagaagcc tttccccttt actattccgg gcagggatgc
     1741 cacccatatg gtcatcagtc tccatggtac caccccgtcg agcctctccc tggatggatt
     1801 ccatggctgt ggaatacaga gccttttggg gggccggtct tcgagtatca actgagtact
     1861 gtgtttcact gcttaagggc tctcgttgtg catgatctat attatggatt gacatcaaga
     1921 ggctgtaatc cattatcttg aagctctgca gcaccaaaca gtcacgctgc agggtcttac
     1981 agagagcgtt gtacatgtca gcatccaaaa aaagaccatc agggatgtct tgtaagaagt
     2041 ctaggtcttt aaatgtggga agaggcttct ctcgctcttt ctgggaagcc cgccgtttgt
     2101 aggttgagcc tttgaggtca tatttgatat gcatttttac cgatcttggt aaaagattgt
     2161 tcatcaccac aatccgaatg ttcttgccac ctgcctgcac acagtacagt ccatagaatt
     2221 taggcagcaa agtccgaggg ttctggttga ggttcatgta gtatcctgga agcagcttct
     2281 gcagaaattc cgcctcttta tgttggactg tcttaataat gaactcatcg tcgctggaca
     2341 catagaatag ggaaccacta gctccagagc tacagagttc aatcagcggc tcactgcaga
     2401 gggaatacaa gtaatcatcg ggccggatac caaatagctc ccggaagtag cggaaggcaa
     2461 caggtgcata ggtcttgaaa cgaaagtcat tgtagtgatg agcaggggtc aggttgctcc
     2521 cttcactggg aaagaagata ctctcaacca cgtagaaatc ttgcatgagg acatcacgct
     2581 ctggtttggt actcaggctc cccacagtgt gggtaatgcc taactggatg gcacctttca
     2641 aggctgatga ggttgtcttt ttatatgttg tctctcctga ggaatcaaca cttctatggc
     2701 ctattttctt gatgggcatg ccagaggcat aaggcaccaa tgagatgtaa gaatcctgtc
     2761 tagcttccaa gacctcagat gccatgggtc tcttgattcc agatgctgct gaggacaagg
     2821 tacaggaagg gaccgcggga tcaaaggatg aaaaaccgac cgaagacgac ggcccggagg
     2881 aggccgacgc catcgcggta ccgtcgactg cagaattcga agcttgagca ctaccaccag
     2941 cactaccacc ctttgagatt cgtcggaaca catgataata gaggtcccag gcttgggtga
     3001 ggtccttgac attccctgat ttcatgtact tcctgcacca ctcttgggcc tccattaaat
     3061 ctcgaccata ggcctgatta aaggatgttt ccttcagagt ctgggggccc cgttccatca
     3121 tagcatgcaa gggctccagc acctcaaaca tgcctttcac gttcctttcc ccaaagtaca
     3181 aacgagatgc ctcttccagg ccttcatgcc acatctcatg ccagaggatt ctagatagga
     3241 tagcaccagc acctccagct cgagatctga gtccggactc ttccatcacg ccgatctgcc
     3301 aggccagaac ctgggcgact agctcgcagg cttggcggcc atcgagatcg acgtagtacg
     3361 gcgttcggtg atggcaaacg atcaatcccc acagtcgctc gccgcgcaaa atcgagatcg
     3421 acatcgtgcc gtgcatgcct atgttgcgca tgaattccag atggacgggc gagacgctgc
     3481 gcaggatggc gaagctaaga tcaatcggcc gcccggtgac cggattgagg tctggggtga
     3541 ccggcaccgg ccgataattg atatcgggaa tgatccgtac cgggttgatg gtatagagcc
     3601 gacgggcctg cgccggcacg gttgaggcag gatagtgcag gcctagtttt gactcgacct
     3661 cggcgcaccg atcctctgcg atcacttcgc cgctgaagtc ggaggcgaag cgatagatca
     3721 tcacccgatc gaagccggta atcttccgca cctcttgcgc cgcggcggcg caggcgcttt
     3781 ccaaggtttc ggcggcctgc aggcggcgga tggcgctgtt ggtgcggcgg aagaacgcct
     3841 gcggctcggc gacgtcccgc tggggaggct cgagctcgag gaagatgagc tgatcatggc
     3901 gatgccagga gccgatgaag cctgcgtcct ttcgcatcgt gaagccgaca gtgatcggtg
     3961 ctccgacggc cgccccgggc tcggccaagg cgatcgtcag acggttgtgc gtctccgagt
     4021 cgaagacatc ggccgcagag cggccgatca gggcgccgat cgccagtccg gtgagttcgg
     4081 gaaggttgtc gctgccggca acgatcgtca tgtcggcggc gagggcgagc agcagtccat
     4141 gcggttggat ggcaccgggg atatggatcg gctcatcgtc gcaggtcaag aggtcaggct
     4201 gcctggcgac ggatccttcc gccatggtgg cgaccggtag cgctagcgga tctgacggtt
     4261 cactaaacca gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     4321 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     4381 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     4441 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     4501 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     4561 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     4621 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     4681 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     4741 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     4801 aactaatgac cccgtaattg attactatta ataactaatg catggcggta atacggttat
     4861 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4921 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4981 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5281 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5341 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5401 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5461 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5521 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5581 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5641 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaacctg
     5701 aggctatggc agggcctgcc gccccgacgt tggctgcgag ccctgggcct tcacccgaac
     5761 ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg
     5821 gtggggtatc gacagagtgc cagccctggg accgaacccc gcgtttatga acaaacgacc
     5881 caacaccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt ccttccggta
     5941 ttgtctcctt ccgtgtttca gttagcctcc ccctagggtg ggcgaagaac tccagcatga
     6001 gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc
     6061 tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
     6121 cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc
     6181 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     6241 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     6301 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     6361 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt
     6421 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     6481 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     6541 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     6601 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     6661 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     6721 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
     6781 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     6841 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     6901 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     6961 tgtctcttga tcgatct
//
