LOCUS       sequence-399754-        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"
     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..1560
                     /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"
     CDS             1609..1887
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     polyA_signal    3765..3886
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3802..3821)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3856..3875
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3893..4348)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4030..4051)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4242..4261
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4375..4479
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4481..4838
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4481..4672
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4504..4524)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4689..4824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4873..5667
                     /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             4873..5667
                     /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(4927..4946)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5537..5556
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5855..5874)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5899..5946
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6275..6863
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6764..6783
                     /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 ccatggcgga aggatccgtc gccaggcagc ctgacctctt gacctgcgac
      661 gatgagccga tccatatccc cggtgccatc caaccgcatg gactgctgct cgccctcgcc
      721 gccgacatga cgatcgttgc cggcagcgac aaccttcccg aactcaccgg actggcgatc
      781 ggcgccctga tcggccgctc tgcggccgat gtcttcgact cggagacgca caaccgtctg
      841 acgatcgcct tggccgagcc cggggcggcc gtcggagcac cgatcactgt cggcttcacg
      901 atgcgaaagg acgcaggctt catcggctcc tggcatcgcc atgatcagct catcttcctc
      961 gagctcgagc ctccccagcg ggacgtcgcc gagccgcagg cgttcttccg ccgcaccaac
     1021 agcgccatcc gccgcctgca ggccgccgaa accttggaaa gcgcctgcgc cgccgcggcg
     1081 caagaggtgc ggaagattac cggcttcgat cgggtgatga tctatcgctt cgcctccgac
     1141 ttcagcggcg aagtgatcgc agaggatcgg tgcgccgagg tcgagtcaaa actaggcctg
     1201 cactatcctg cctcaaccgt gccggcgcag gcccgtcggc tctataccat caacccggta
     1261 cggatcattc ccgatatcaa ttatcggccg gtgccggtca ccccagacct caatccggtc
     1321 accgggcggc cgattgatct tagcttcgcc atcctgcgca gcgtctcgcc cgtccatctg
     1381 gaattcatgc gcaacatagg catgcacggc acgatgtcga tctcgatttt gcgcggcgag
     1441 cgactgtggg gattgatcgt ttgccatcac cgaacgccgt actacgtcga tctcgatggc
     1501 cgccaagcct gcgagctagt cgcccaggtt ctggcctggc agatcggcgt gatggaagag
     1561 tccggactca gatctcgagc tggaggtgct ggtgctatcc tatctagaat cctctggcat
     1621 gagatgtggc atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg
     1681 aaaggcatgt ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact
     1741 ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg
     1801 tgcaggaagt acatgaaatc agggaatgtc aaggacctca cccaagcctg ggacctctat
     1861 tatcatgtgt tccgacgaat ctcaaagggt ggtagtgctg gtggtagtgc tcaagcttcg
     1921 aattctgcag tcgacggtac cgcgatggcg tcggcctcct ccgggccgtc gtcttcggtc
     1981 ggtttttcat cctttgatcc cgcggtccct tcctgtacct tgtcctcagc agcatctgga
     2041 atcaagagac ccatggcatc tgaggtcttg gaagctagac aggattctta catctcattg
     2101 gtgccttatg cctctggcat gcccatcaag aaaataggcc atagaagtgt tgattcctca
     2161 ggagagacaa catataaaaa gacaacctca tcagccttga aaggtgccat ccagttaggc
     2221 attacccaca ctgtggggag cctgagtacc aaaccagagc gtgatgtcct catgcaagat
     2281 ttctacgtgg ttgagagtat cttctttccc agtgaaggga gcaacctgac ccctgctcat
     2341 cactacaatg actttcgttt caagacctat gcacctgttg ccttccgcta cttccgggag
     2401 ctatttggta tccggcccga tgattacttg tattccctct gcagtgagcc gctgattgaa
     2461 ctctgtagct ctggagctag tggttcccta ttctatgtgt ccagcgacga tgagttcatt
     2521 attaagacag tccaacataa agaggcggaa tttctgcaga agctgcttcc aggatactac
     2581 atgaacctca accagaaccc tcggactttg ctgcctaaat tctatggact gtactgtgtg
     2641 caggcaggtg gcaagaacat tcggattgtg gtgatgaaca atcttttacc aagatcggta
     2701 aaaatgcata tcaaatatga cctcaaaggc tcaacctaca aacggcgggc ttcccagaaa
     2761 gagcgagaga agcctcttcc cacatttaaa gacctagact tcttacaaga catccctgat
     2821 ggtctttttt tggatgctga catgtacaac gctctctgta agaccctgca gcgtgactgt
     2881 ttggtgctgc agagcttcaa gataatggat tacagcctct tgatgtcaat ccataatata
     2941 gatcatgcac aacgagagcc cttaagcagt gaaacacagt actcagttga tactcgaaga
     3001 ccggcccccc aaaaggctct gtattccaca gccatggaat ccatccaggg agaggctcga
     3061 cggggtggta ccatggagac tgatgaccat atgggtggca tccctgcccg gaatagtaaa
     3121 ggggaaaggc ttctgcttta tattggcatc attgacattc tacagtctta caggtttgtt
     3181 aagaagttgg agcactcttg gaaagccctg gtacatgacg gagacactgt ctcagtgcat
     3241 cgcccaggct tctacgctga acggttccag cgcttcatgt gcaacacagt atttaagaag
     3301 attcccttga agccttctcc ttccaaaaag tttcggtctg gctcatcttt ctctcggcga
     3361 gcaggctcca gtggcaactc ctgcattact taccagccat cggtctctgg ggaacacaag
     3421 gcacaagtga caacaaaggc agaagtggag ccaggcgttc accttggtcg tcctgatgtt
     3481 ttacctcaga ctccaccttt ggaggaaatc agtgagggct cgcctattcc tgaccccagt
     3541 ttctcacctc tagttggaga gactttgcaa atgctaacta caagtacaac cttggaaaag
     3601 cttgaagttg cagagtcaga gttcacccat taacgggatc caccggatct agataactga
     3661 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     3721 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     3781 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3841 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     3901 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3961 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     4021 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     4081 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     4141 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     4201 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     4261 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     4321 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4381 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4441 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     4501 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     4561 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4621 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4681 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4741 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4801 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     4861 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4921 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4981 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     5041 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5101 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5161 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5221 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5281 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5341 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5401 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5461 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5521 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5581 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5641 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5701 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5761 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5821 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     5881 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5941 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     6001 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     6061 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     6121 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6181 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6241 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6301 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6361 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6421 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6481 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6541 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6601 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6661 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6721 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6781 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6841 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6901 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6961 gtattaccgc catgcat
//
