LOCUS       sequence-427397-        6351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For testing localization of pFAST and iRFP670 proteins to 
            mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6351)
  TITLE     Tebo lab plasmids 2024
REFERENCE   2  (bases 1 to 6351)
  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"
COMMENT     Sequence Label: Mito-FAST-IRES-iRFP670
FEATURES             Location/Qualifiers
     source          1..6351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             122..916
                     /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             122..916
                     /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(176..195)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     786..805
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1104..1123)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1148..1195
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1524..2112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2013..2032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2287..2590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2591..2794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2745..2765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2823..2897
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      2925..2934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2931..3305
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLANMDDEQLDRLAFGVIQLDGDGNILLYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDTPEFYGKFKEGAASGNLNTMFEWTIPTSRGPTK
                     VKVHLKKALSGDRYWVFVKRV"
     CDS             3312..3341
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3351..3374
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3386..3960,3961..3963,3964..3972)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3553..3570)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3780..3799
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4438..5367
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             5374..5403
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    5437..5558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5474..5493)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5528..5547
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5565..6020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5702..5723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5914..5933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6047..6151
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6176..6196)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6220..6351,1..79)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        join(6242..6351,1..87)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      join(6289..6351,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
      121 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
      181 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
      241 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
      301 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
      361 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
      421 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
      481 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
      541 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
      601 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
      661 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
      721 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
      781 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
      841 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
      901 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
      961 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     1021 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     1081 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     1141 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     1201 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     1261 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     1321 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     1381 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     1441 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1501 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1561 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1621 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     1681 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1741 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1801 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1861 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     1921 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1981 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2041 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2101 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2161 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2221 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     2281 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     2341 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     2401 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     2461 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     2521 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     2581 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     2641 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     2701 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     2761 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
     2821 gcatgtccgt cctgacgccg ctgctgctgc ggggcttgac aggctcggcc cggcggctcc
     2881 cagtgccgcg cgccaagatc cattcgttgg gggatccacc ggtcgccacc atggagcatg
     2941 ttgcctttgg cagtgaggac atcgagaaca ctctggccaa tatggacgac gaacaactgg
     3001 ataggttggc ctttggcgta attcagctcg atggtgacgg gaatatcctg ctgtacaatg
     3061 ctgctgaagg ggacatcact ggcagagatc ccaaacaggt gattgggaag aacttcttca
     3121 aggatgttgc acctggaacg gatactcccg agttttacgg caaattcaag gaaggcgcag
     3181 cgtcagggaa tctgaacacc atgttcgaat ggacgatacc gacaagcagg ggaccaacca
     3241 aggtcaaggt gcacttgaag aaagcccttt ccggtgacag atattgggtc tttgtgaaac
     3301 gggtgggatc cgaacaaaag cttatttctg aagaggactt gtaactcgag gactacaagg
     3361 acgacgacga caagcccggg atccgcccct ctccctcccc cccccctaac gttactggcc
     3421 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3481 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3541 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     3601 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     3661 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     3721 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     3781 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     3841 tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa
     3901 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     3961 atggccacaa ccatggtggg tcggaacagc gccatcgccg ccggtgtatg cggggccctt
     4021 ttcattgggt actgcatcta cttcgaccgc aaaagacgaa gtgaccccaa cttcaagaac
     4081 aggcttcgag aacgaagaaa gaaacagaag cttgccaagg agagagctgg gctttccaag
     4141 ttacctgacc ttaaagatgc tgaagctgtt cagaagttct tccttgaaga aatacagctt
     4201 ggtgaagagt tactagctca aggtgaatat gagaagggcg tagaccatct gacaaatgca
     4261 attgctgtgt gtggacagcc acagcagtta ctgcaggtct tacagcaaac tcttccacca
     4321 ccagtgttcc agatgcttct gactaagctc ccaacaatta gtcagagaat tgtaagtgct
     4381 cagagcttgg ctgaagatga tgtggaaggc ggtagcgggg atccaccggt cgctactgcg
     4441 cgtaaggtcg atctcacctc ctgcgatcgc gagccgatcc acatccccgg cagcattcag
     4501 ccgtgcggct gcctgctagc ctgcgacgcg caggcggtgc ggatcacgcg cattacggaa
     4561 aatgccggcg cgttctttgg acgcgaaact ccgcgggtcg gtgagctact cgccgattac
     4621 ttcggcgaga ccgaagccca tgcgctgcgc aacgcactgg cgcagtcctc cgatccaaag
     4681 cgaccggcgc tgatcttcgg ttggcgcgac ggcctgaccg gccgcacctt cgacatctca
     4741 ctgcatcgcc atgacggtac atcgatcatc gagttcgagc ctgcggcggc cgaacaggcc
     4801 gacaatccgc tgcggctgac gcggcagatc atcgcgcgca ccaaagaact gaagtcgctc
     4861 gaagagatgg ccgcacgggt gccgcgctat ctgcaggcga tgctcggcta tcaccgcgtg
     4921 atgttgtacc gcttcgcgga cgacggctcc gggatggtga tcggcgaggc gaagcgcagc
     4981 gacctcgaga gctttctcgg tcagcacttt ccggcgtcgc tggtcccgca gcaggcgcgg
     5041 ctactgtact tgaagaacgc gatccgcgtg gtctcggatt cgcgcggcat cagcagccgg
     5101 atcgtgcccg agcacgacgc ctccggcgcc gcgctcgatc tgtcgttcgc gcacctgcgc
     5161 agcatctcgc cctgccatct cgaatttctg cggaacatgg gcgtcagcgc ctcgatgtcg
     5221 ctgtcgatca tcattgacgg cacgctatgg ggattgatca tctgtcatca ttacgagccg
     5281 cgtgccgtgc cgatggcgca gcgcgtcgcg gccgaaatgt tcgccgactt cttatcgctg
     5341 cacttcaccg ccgcccacca ccaacgcgga tccgaacaaa agcttatttc tgaagaggac
     5401 ttgtgattaa ttaagcaagg gcgaattgtt gttgttaact tgtttattgc agcttataat
     5461 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5521 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     5581 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5641 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5701 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5761 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5821 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5881 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5941 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6001 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6061 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6121 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     6181 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     6241 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     6301 actccgccca gttccgccca ttctccgccc catggctgac taattttttt t
//
