LOCUS       sequence-219818-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cytosolic iRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5007)
  AUTHORS   Goulden BD, Pacheco J, Dull A, Zewe JP, Deiters A, Hammond GRV
  TITLE     A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is 
            predominantly a class I PI3K signaling product.
  JOURNAL   J Cell Biol. 2019 Mar 4;218(3):1066-1079. doi: 
            10.1083/jcb.201809026. Epub 2018 Dec 27.
  PUBMED    30591513
REFERENCE   2  (bases 1 to 5007)
  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: 
            "10.1083/jcb.201809026"; journalName: "J Cell Biol"; date: 
            "2019-03-4- 4"; volume: "218"; issue: "3"; pages: "1066-1079"
COMMENT     Sequence Label: pNES-iRFP-C1
FEATURES             Location/Qualifiers
     source          1..5007
                     /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      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             658..1605
                     /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"
     misc_feature    1606..1671
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1795..1916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1832..1851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1886..1905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1923..2378)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2060..2081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2272..2291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2405..2509
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2511..2868
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2511..2702
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2534..2554)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2719..2854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2903..3697
                     /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             2903..3697
                     /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(2957..2976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3567..3586
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3885..3904)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3929..3976
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4305..4893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4794..4813
                     /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 gctagccacc
      601 atggctctgc agaaaaagtt ggaagagctt gagctggatg aggcaccggt cgccaccatg
      661 gcggaaggat ccgtcgccag gcagcctgac ctcttgacct gcgacgatga gccgatccat
      721 atccccggtg ccatccaacc gcatggactg ctgctcgccc tcgccgccga catgacgatc
      781 gttgccggca gcgacaacct tcccgaactc accggactgg cgatcggcgc cctgatcggc
      841 cgctctgcgg ccgatgtctt cgactcggag acgcacaacc gtctgacgat cgccttggcc
      901 gagcccgggg cggccgtcgg agcaccgatc actgtcggct tcacgatgcg aaaggacgca
      961 ggcttcatcg gctcctggca tcgccatgat cagctcatct tcctcgagct cgagcctccc
     1021 cagcgggacg tcgccgagcc gcaggcgttc ttccgccgca ccaacagcgc catccgccgc
     1081 ctgcaggccg ccgaaacctt ggaaagcgcc tgcgccgccg cggcgcaaga ggtgcggaag
     1141 attaccggct tcgatcgggt gatgatctat cgcttcgcct ccgacttcag cggcgaagtg
     1201 atcgcagagg atcggtgcgc cgaggtcgag tcaaaactag gcctgcacta tcctgcctca
     1261 accgtgccgg cgcaggcccg tcggctctat accatcaacc cggtacggat cattcccgat
     1321 atcaattatc ggccggtgcc ggtcacccca gacctcaatc cggtcaccgg gcggccgatt
     1381 gatcttagct tcgccatcct gcgcagcgtc tcgcccgtcc atctggaatt catgcgcaac
     1441 ataggcatgc acggcacgat gtcgatctcg attttgcgcg gcgagcgact gtggggattg
     1501 atcgtttgcc atcaccgaac gccgtactac gtcgatctcg atggccgcca agcctgcgag
     1561 ctagtcgccc aggttctggc ctggcagatc ggcgtgatgg aagagtccgg actcagatct
     1621 cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc caccggatct
     1681 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     1741 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     1801 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1861 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     1921 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     1981 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2041 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2101 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2161 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2221 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2281 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2341 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     2401 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     2521 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2581 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2701 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2761 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2821 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     2881 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     2941 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3001 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3061 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     3121 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3181 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3241 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3301 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3361 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3421 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3481 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3541 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3601 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3661 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3721 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3781 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3841 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     3901 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     3961 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4021 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     4081 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     4141 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4201 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4261 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4321 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4381 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4441 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4501 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4561 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4621 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4681 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4741 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4801 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4861 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4921 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4981 gtggataacc gtattaccgc catgcat
//
