LOCUS       sequence-224782-        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"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      231..240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      288..297
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             294..1241
                     /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    1242..1307
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1431..1552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1468..1487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1522..1541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1559..2014)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1696..1717)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1908..1927
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2041..2145
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2147..2504
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2147..2338
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2170..2190)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2355..2490
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2539..3333
                     /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             2539..3333
                     /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(2593..2612)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3203..3222
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3521..3540)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3565..3612
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3941..4529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4430..4449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4704..5007
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gccaccatgg
      241 ctctgcagaa aaagttggaa gagcttgagc tggatgaggc accggtcgcc accatggcgg
      301 aaggatccgt cgccaggcag cctgacctct tgacctgcga cgatgagccg atccatatcc
      361 ccggtgccat ccaaccgcat ggactgctgc tcgccctcgc cgccgacatg acgatcgttg
      421 ccggcagcga caaccttccc gaactcaccg gactggcgat cggcgccctg atcggccgct
      481 ctgcggccga tgtcttcgac tcggagacgc acaaccgtct gacgatcgcc ttggccgagc
      541 ccggggcggc cgtcggagca ccgatcactg tcggcttcac gatgcgaaag gacgcaggct
      601 tcatcggctc ctggcatcgc catgatcagc tcatcttcct cgagctcgag cctccccagc
      661 gggacgtcgc cgagccgcag gcgttcttcc gccgcaccaa cagcgccatc cgccgcctgc
      721 aggccgccga aaccttggaa agcgcctgcg ccgccgcggc gcaagaggtg cggaagatta
      781 ccggcttcga tcgggtgatg atctatcgct tcgcctccga cttcagcggc gaagtgatcg
      841 cagaggatcg gtgcgccgag gtcgagtcaa aactaggcct gcactatcct gcctcaaccg
      901 tgccggcgca ggcccgtcgg ctctatacca tcaacccggt acggatcatt cccgatatca
      961 attatcggcc ggtgccggtc accccagacc tcaatccggt caccgggcgg ccgattgatc
     1021 ttagcttcgc catcctgcgc agcgtctcgc ccgtccatct ggaattcatg cgcaacatag
     1081 gcatgcacgg cacgatgtcg atctcgattt tgcgcggcga gcgactgtgg ggattgatcg
     1141 tttgccatca ccgaacgccg tactacgtcg atctcgatgg ccgccaagcc tgcgagctag
     1201 tcgcccaggt tctggcctgg cagatcggcg tgatggaaga gtccggactc agatctcgag
     1261 ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc ggatctagat
     1321 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     1381 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     1441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa
     1561 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1621 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     1681 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1741 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     1801 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     1861 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     1921 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     1981 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     2041 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2101 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     2161 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2221 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2281 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2341 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2401 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     2461 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     2521 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2581 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2641 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2701 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     2761 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2821 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2881 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2941 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3001 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3061 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3121 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3181 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3241 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3301 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3361 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3421 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3481 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     3541 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     3601 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     3661 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     3721 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     3781 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3841 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4081 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4621 ataaccgtat taccgccatg cattagttat taatagtaat caattacggg gtcattagtt
     4681 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     4741 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     4801 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     4861 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     4921 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     4981 tacgtattag tcatcgctat taccatg
//
