LOCUS       sequence-287354-        5581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal half of split PI-PLC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5581)
  AUTHORS   Zewe JP, Miller AM, Sangappa S, Wills RC, Goulden BD, Hammond GRV
  TITLE     Probing the subcellular distribution of phosphatidylinositol reveals
            a surprising lack at the plasma membrane.
  JOURNAL   J Cell Biol. 2020 Mar 2;219(3). pii: 133808. doi: 
            10.1083/jcb.201906127.
  PUBMED    32211893
REFERENCE   2  (bases 1 to 5581)
  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 
            Biol."; date: "2020-03-2"; volume: "219(3). pii: 133808. doi"; 
            pages: " 10.1083/jcb.201906127"
COMMENT     Sequence Label: iRFP-N1-PI-PLCC100-FRB
FEATURES             Location/Qualifiers
     source          1..5581
                     /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"
     CDS             complement(940..957)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             972..1250
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1257..1283
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1290..1299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1296..2246
                     /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"
     polyA_signal    2369..2490
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2406..2425)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2460..2479
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2497..2952)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2634..2655)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2846..2865
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2979..3083
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3085..3442
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3085..3276
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3108..3128)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3293..3428
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3477..4271
                     /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             3477..4271
                     /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(3531..3550)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4141..4160
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4459..4478)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4503..4550
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4879..5467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5368..5387
                     /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 gctagcacca
      601 tgggcgcacc taaccaagta attcaagatg actacaatgg tccgagtgtg aaaacaaaat
      661 tcaaagagat tgtccagact gcttatcaag cttccaaagc ggacaataaa ctttttctta
      721 accatattag cgccacttca ttaacattca cacctcgtca gtatgctgca gcattaaaca
      781 acaaagtaga gcaattcgta ctcaacttaa catcggaaaa agttcgagga ttaggcatac
      841 taatcatgga cttccccgaa aaacaaacaa ttaaaaacat cataaaaaac aataaattca
      901 acatcggtac cgcgggcccg agatctgcga attcaggtgc tggtgctggt gctggtgcta
      961 tcctatctag aatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
     1021 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
     1081 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
     1141 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
     1201 tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag actagttatc
     1261 cgtacgacgt accagactac gcaccggtcg ccaccatggc ggaaggatcc gtcgccaggc
     1321 agcctgacct cttgacctgc gacgatgagc cgatccatat ccccggtgcc atccaaccgc
     1381 atggactgct gctcgccctc gccgccgaca tgacgatcgt tgccggcagc gacaaccttc
     1441 ccgaactcac cggactggcg atcggcgccc tgatcggccg ctctgcggcc gatgtcttcg
     1501 actcggagac gcacaaccgt ctgacgatcg ccttggccga gcccggggcg gccgtcggag
     1561 caccgatcac tgtcggcttc acgatgcgaa aggacgcagg cttcatcggc tcctggcatc
     1621 gccatgatca gctcatcttc ctcgagctcg agcctcccca gcgggacgtc gccgagccgc
     1681 aggcgttctt ccgccgcacc aacagcgcca tccgccgcct gcaggccgcc gaaaccttgg
     1741 aaagcgcctg cgccgccgcg gcgcaagagg tgcggaagat taccggcttc gatcgggtga
     1801 tgatctatcg cttcgcctcc gacttcagcg gcgaagtgat cgcagaggat cggtgcgccg
     1861 aggtcgagtc aaaactaggc ctgcactatc ctgcctcaac cgtgccggcg caggcccgtc
     1921 ggctctatac catcaacccg gtacggatca ttcccgatat caattatcgg ccggtgccgg
     1981 tcaccccaga cctcaatccg gtcaccgggc ggccgattga tcttagcttc gccatcctgc
     2041 gcagcgtctc gcccgtccat ctggaattca tgcgcaacat aggcatgcac ggcacgatgt
     2101 cgatctcgat tttgcgcggc gagcgactgt ggggattgat cgtttgccat caccgaacgc
     2161 cgtactacgt cgatctcgat ggccgccaag cctgcgagct agtcgcccag gttctggcct
     2221 ggcagatcgg cgtgatggaa gagtgaagcg gccgcgactc tagatcataa tcagccatac
     2281 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2341 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2401 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2461 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     2521 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2581 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2641 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2701 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2761 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2821 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2881 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2941 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3001 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3061 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3121 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3181 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3241 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3301 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3361 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3421 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3481 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3541 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3601 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3661 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3721 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3781 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3841 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3901 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3961 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4021 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4081 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4141 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4201 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4261 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4321 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4381 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4441 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4501 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4561 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     4621 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4681 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4741 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4801 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4861 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4921 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4981 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5041 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5101 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5161 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5221 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5281 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5341 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5401 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5461 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5521 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     5581 t
//
