LOCUS       sequence-434128-        5388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB targeted to the mitochondria with the tail of the mitochondrial 
            outer membrane protein Fis1 with an iRFP fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5388)
  AUTHORS   Doyle CP, Rectenwald A, Timple L, Hammond GRV
  TITLE     Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a 
            Major Player in PM PI4P Turnover.
  JOURNAL   Contact (Thousand Oaks). 2024 Feb 7;7:25152564241229272. doi: 
            10.1177/25152564241229272. eCollection 2024 Jan-Dec.
  PUBMED    38327560
REFERENCE   2  (bases 1 to 5388)
  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: "Contact 
            (Thousand Oaks)."; date: "2024-02-7"; volume: "7:25152564241229272. 
            doi"; pages: " 10.1177/25152564241229272. eCollection 2024 Jan-Dec"
COMMENT     Sequence Label: iRFP713-FRB-Fis1tail
FEATURES             Location/Qualifiers
     source          1..5388
                     /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    2176..2297
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2213..2232)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2267..2286
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2304..2759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2441..2462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2653..2672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2786..2890
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2892..3249
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2892..3083
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2915..2935)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3100..3235
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3284..4078
                     /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             3284..4078
                     /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(3338..3357)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3948..3967
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4266..4285)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4310..4357
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4686..5274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5175..5194
                     /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 cgcgggactc gtgggcatgg ccatcgtggg aggcatggcc
     1981 ctgggtgtgg cgggactggc cggactcatc ggacttgctg tgtccaagtc caaatcctga
     2041 ggcccgggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     2101 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     2161 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2221 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2281 ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     2341 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2401 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2461 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2521 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2581 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2641 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2701 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2821 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2881 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2941 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3001 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3061 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3121 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3181 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3241 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3301 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3361 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3421 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3481 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3541 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3601 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3661 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3721 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3781 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3841 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3901 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3961 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4021 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     4081 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4141 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4201 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     4261 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     4321 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     4381 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     4441 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     4501 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     4561 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4621 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4681 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4741 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4801 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4861 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4921 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4981 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5041 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5101 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5161 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5221 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5281 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5341 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
