LOCUS       sequence-139728-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dL5(E52D)-mCer3 fusion protein in mitochondria of 
            mammalian cells. (MBIC5, dL5**, FAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  AUTHORS   Telmer CA, Verma R, Teng H, Andreko S, Law L, Bruchez MP
  TITLE     Rapid, specific, no-wash, far-red fluorogen activation in 
            subcellular compartments by targeted fluorogen activating proteins.
  JOURNAL   ACS Chem Biol. 2015 May 15;10(5):1239-46. doi: 10.1021/cb500957k. 
            Epub 2015 Feb 16.
  PUBMED    25650487
REFERENCE   2  (bases 1 to 7104)
  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: "10.1021/cb500957k";
            journalName: "ACS Chem Biol"; date: "2015-05-15- 15"; volume: "10"; 
            issue: "5"; pages: "1239-46"
COMMENT     Sequence Label: pcDNA3.1-COXIV-COX8-dL5-2XG4S-mCer3
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     867..886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        867..885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1026..1100
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1152..1871
                     /codon_start=1
                     /product="fluorogen-activating protein composed of a dimer 
                     of the L5-MG single-chain antibody fragment (Szent-Gyorgyi 
                     et al., 2008)"
                     /label=FAP-alpha-2
                     /note="binds malachite green derivatives to produce red 
                     fluorescence"
                     /translation="QAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGHYANWFQQKPGQA
                     PRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGT
                     QLTVLSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGH
                     YANWFQQKPGQAPRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSL
                     SDVDGYLFGGGTQLTVLS"
     CDS             join(1929..1931,1932..1934,1935..2648)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1974..1995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2235..2254)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2582..2603
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2698..2715)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2704..2928
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2974..3402
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3061..3080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3271..3292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3411..3431)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3416..3745
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3596..3731
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3658..3677
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3812..4606
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3866..3885)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4476..4495
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4780..4901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4817..4836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4871..4890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4950..4966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4966)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4963..4985)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4974..4990
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4998..5004,5005..5022,5023..5028))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5043..5064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5181..5198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5352..5937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5432..5451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6108..6899,6900..6968))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6731..6750
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6969..7073)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      841 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctggc
      901 tagcgtttaa acttaagctt gccaccggat ccgccaccat gctcgctaca agagttttct
      961 cacttgtcgg caaaagagcc atttctacct cggtttgtgt aagggctgaa gaatctagag
     1021 aattcatgtc cgtcctgacg ccgctgctgc tgcggggctt gacaggctcg gcccggcggc
     1081 tcccagtgcc gcgcgccaag atccattcgt tgccgccgga ggggaagctt gaattcggcc
     1141 tttctggcct gcaggccgtc gttacccaag aacctagtgt taccgttagc ccaggtggta
     1201 ctgttatact tacttgtgga agtggtacgg gtgccgtcac atctggtcat tatgcaaatt
     1261 ggtttcaaca aaaaccagga caagctccaa gagctttgat ttttgatact gataagaagt
     1321 attcttggac cccaggtaga ttttctggat ctttgctggg agcaaaggca gctttgacaa
     1381 tatcagatgc tcagcctgag gacgaagccg agtattactg ttctcttagc gacgtggatg
     1441 gctacttgtt tggcggtgga acacaactga cggttctgtc cggtggtggc ggctctggtg
     1501 gcggtggcag cggcggtggt ggttccggag gcggcggttc tcaggctgtg gtgactcagg
     1561 agccgtcagt gactgtgtcc ccaggaggga cagtcattct cacttgtggc tccggcactg
     1621 gagctgtcac cagtggtcat tatgccaact ggttccagca gaagcctggc caagccccca
     1681 gggcacttat atttgacacc gacaagaagt attcctggac ccctggccga ttctcaggct
     1741 ccctccttgg ggccaaggct gccctgacca tctcggatgc gcagcctgaa gatgaggctg
     1801 agtattactg ttcgctctcc gacgttgacg gttatctgtt cggaggaggc acccagctga
     1861 ccgtcctctc cgcggccggt ctggcccata tgggaggcgg tgggtctggt ggcggtggca
     1921 gcaccggtat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1981 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2041 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2101 ggcccaccct cgtgaccacc ctgagctggg gcgtgcagtg cttcgcccgc taccccgacc
     2161 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2221 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2281 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2341 tggggcacaa gctggagtac aacgccatcc acggcaacgt ctatatcacc gccgacaagc
     2401 agaagaacgg catcaaggcc aacttcggcc tcaactgcaa catcgaggac ggcagcgtgc
     2461 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2521 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     2581 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2641 acaagtaact cgaggcagct gtcgactcta gagggcccgt ttaaacccgc tgatcagcct
     2701 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2761 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2821 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2881 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     2941 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3001 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3061 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3121 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3181 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3241 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3301 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3361 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     3421 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3481 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3541 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3601 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3661 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     3721 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     3781 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3841 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3901 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3961 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     4021 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4081 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4141 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4201 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4261 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4321 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4381 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4441 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4501 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4561 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4621 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4681 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4741 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4801 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4861 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4921 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4981 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5041 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5101 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5161 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5221 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5281 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5341 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5401 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5461 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5521 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5581 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5641 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5701 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5761 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5821 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5881 ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5941 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6001 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6061 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6121 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6181 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6241 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6301 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6361 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6421 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6481 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6541 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6601 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6661 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6721 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6781 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6841 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6901 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6961 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7021 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7081 ccccgaaaag tgccacctga cgtc
//
