LOCUS       sequence-358087-        9500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression construct encoding shRNA-resistant mtIF3 and 
            mito-mTFP1 from separate promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9500)
  AUTHORS   Lee S, Park D, Lim C, Kim JI, Min KT
  TITLE     mtIF3 is locally translated in axons and regulates mitochondrial 
            translation for axonal growth.
  JOURNAL   BMC Biol. 2022 Jan 7;20(1):12. doi: 10.1186/s12915-021-01215-w.
  PUBMED    34996455
REFERENCE   2  (bases 1 to 9500)
  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: "BMC Biol.";
            date: "2022-01-7"; volume: "20(1):12. doi"; pages: " 
            10.1186/s12915-021-01215-w"
COMMENT     Sequence Label: 
            psiCheck2-SV40p-5'UTR-mtIF3(SR)-3xFLAG-3'UTR-CMVp-mito-mTFP1
FEATURES             Location/Qualifiers
     source          1..9500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        62..253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(85..105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      270..405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          489..621
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     604..623
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1766..1831
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5869..5886
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    5907..5955
                     /note="synthetic polyadenylation signal"
     enhancer        5962..6265
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6266..6469
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6420..6440
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             6498..6572
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             6594..6665
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      6693..6702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6699..7409
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     polyA_signal    7455..7576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7466..7485)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7520..7539
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        7709..7813
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7814..7882,7883..8674)
                     /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     complement(8032..8051)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8845..9433
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9334..9353
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcg gaagccgacg cggtgctccg gcggagtcca
      721 ggtacggacc tgctagttct tcctgctcgc ctaccccggc ccagggtctc actatttatc
      781 tctggctgtc ctgaaacgca ctcggtagac cagtctgacc tcaaacctat agaaatcctc
      841 ctgcctctac ctccgaagtg ctgggattaa aggcgtgcac cgctcgccgt gtcaggactt
      901 ttgaaattcc ttccggaatt gaaggggtac catggctgtt cttctgatga ggttgatgct
      961 acaaactaca aagcttgacc ataacttaat tggaagatgt ttgcaaagac acgctgtgaa
     1021 gccagatcca gcacagctgt ccctgtctgc gtccacccca aagctgttgt acctgacttc
     1081 tgcaaaaggt tttagtactg ctggagaccc acagggtgag aggaaacaga aaagaaggga
     1141 tgctttctct aacaccggcc ggaagattag tgagcggatt atccgcgtcc tggatgagaa
     1201 gggcatggac ctggggatga tgcatcgagc agatgtgatc agactcatga acaagcagga
     1261 cctgaggctg gtgcagagga acaccagctc agagccaccg gagtaccagc ttatgaccgg
     1321 agagcagatc caccaggagc ggctaaagct ccgggaacag gaaaaggcca aacccaaaac
     1381 tggaccaact atgacgaaag agctcgtttt ctcttcaaat attggacagc acgatttaga
     1441 caccaagagt aaacagatcc agcagtggat agagaagaaa tatcatgtac aggtgaccat
     1501 taagaggagg aaagatgcgg agcagtcaga ggaggaaacg gaggagatat ttaaccagat
     1561 tctccagact atgcctgaca tagcaacctt ctcaagcaga ccaaaagcca tcagaggagg
     1621 gactgcttcc atgtgtgttt ttcgtcactt gagcaagaaa gaagaaaagg cgtacagaga
     1681 atcccaggag agccagagaa gagacacttt gagcaaagac gacgacggaa acagcaaaga
     1741 gtcggatgtt gtgtgtcaga ccggtgacta taaggaccac gacggagact acaaggatca
     1801 tgatattgat tacaaagacg atgacgataa gtaagcggcc gcctttaata ggaaaagtgc
     1861 aggccaggag cctctgctgg ccgccctgaa gtcttcccgc ctagcagaca gtgagtaagg
     1921 cggctggcgg gggcagctgg gcctctgggc tgctgctgga ctctgcgctg gcgttaggca
     1981 aggagtagtc gggctcttgt ttcctcttag gagggaagta cccactgagg cgagaggcca
     2041 agctgcgtct ctcccgaggg gctcttacct tcaggagtga ggaagacaga caactgagtt
     2101 gcccattaca caacggggat ccactccaac agatgagccc ctgatgggag tcacaccctc
     2161 agcaggcaca gacacgcagg atggggtatg cggatggaca gacgccaagc aggcctcaca
     2221 gccgagtgct ggccccagca gctgccactt actttgagct tcatcctctt cttgtcggga
     2281 tcgtgcacga cactgtgcct catgagactc tgctgcagtg caagggaaag aaggacgctt
     2341 gacctgggcc tgcagacgtg gctcagtgct tacagcattc gctgctcgag gaggacacgg
     2401 gtttggttcc cagcacccgt actccagttc cagtaaaaac tcttggtgtc tgctaacctt
     2461 aagatgcagt ggtccagtgt gcaataataa actaccagct gcctttctgt tgtgtagtga
     2521 atctgacctt cacctgactc cctaaccaga gccaactcct tggcagagcg agcgactgtc
     2581 agcctgctga cacgcatggg atttacttct tcccggtcct cctgagaagg gaaagccggc
     2641 tatgcttgaa catccaggct catgctgggg taactgaatc tacctcagct gcgtccctaa
     2701 gtgccagggg gaaatctgac ctctgccctg ctggctatgc tgacagtgct gaccacggga
     2761 cctttgctgt gggctggtct gggttatgtt cagtgacttt gtaatccaca gtaagagctc
     2821 tttagaaacc agctaggctt aggcaaactg ttcagttccc tactaggaac catgttccca
     2881 cgcgatccat gaatcatgcc aagattccag agtctagagg ctgtgcattt gtgctaccca
     2941 cctcctctcc cagaccctag gcgacggtca gaagctccta atcacctcag aacatcctct
     3001 aagtaggaat acaggccccg ggctctccca ctcagcatgg ctagaatccc ccaggagtgg
     3061 ttctcaacct gctactcatg actcctctgg ggtcatatac cagatcgcct gaatatcatt
     3121 atttacaatc cataccagta gcaaaattat agttatgaag tagcaactaa ataatattat
     3181 ggttgggggt ccccacaaca tgaggaactg ttattgggtc ccagtattag gaaggttgag
     3241 aaccactgct ctagaggtcc taaataggcc tgtttagcct cccaacccca gggctcctgc
     3301 atttccaagt tctgcaggct ggacatgaac acaccagcct ggagcgggag acactcactt
     3361 tcattgcgaa tgtcttgcca cagccagcgt gctcacacac aaatgggcgc ttttcctcgt
     3421 ggaaggagag aatgtggctc tgcaggttga acacggttgt gtaggttctt ccgcagcctt
     3481 gccgcggaca gcggtacaca tccctttccg gggcgtgagt cttcatgtgc tgcttaaggt
     3541 aatctctgcg cttgaacatc ctctgacata cattgcaggt tatgtcctct gggaggagaa
     3601 acacacacgt gtaaccagcg tcccatgagt aacccgtctg gtcctatgac tacacacaat
     3661 ctggggactt cacatgggaa atgtcagctt gtctcactgc tatgctggtt tcaaattcct
     3721 caagttcact catggctccc aacatcctga gtcctcgcct gtcaaggagc aggcgtcctc
     3781 ggtgtgataa ctgagcacga ggaagtatgc cctcatagca tctaccttta ccctctgaca
     3841 cagtagcatc tgctaagtcc tgcagggcct cacgaacacc cgggcacagc cgcaccagct
     3901 ctgaggcctg cagggctccc tcgactgtca cagcccagga agccagcact accctgaact
     3961 cgatgcagag cccccttcct ttacagcgca agtgggtatc tcactctctc agagtacacc
     4021 atgcactccc cagagctcag agcacacaca ccatgggctc cccggggttc agagcacacc
     4081 gggctctccc cagggccgat gcgcttccac agctcctgcc taccttacct ttatgggctt
     4141 ctctcatgtg tttcaggagc tctgtccacg tttttcccat gaaagaacat cccttttgac
     4201 acaggtagcc tgttcaaaac aaaaacaaaa caagttaccc acagcctgtt gaatgcccag
     4261 ggcaatccct aatgacagcc aagaaagccc gtgccaagtg cactcccctc tctgaagtgt
     4321 gctgctctgc tgggtgcaat gcagcgggct ccagtggccc ctgctctgaa agctcccatc
     4381 agccagactt ttgcacactt gagtggcaca taaccaggta gcgtaggaac agtatagcac
     4441 cagtggacgt ggcaaagagg atagcggcaa aacacacagc acagtgtctc ttggcataag
     4501 aggcaaacac tcaaataaca caacaggcgc ttgcttgtca cagaactttg tctactcagc
     4561 gtgactacag aatagaccca gcctaccctg ccggtggcat gttcctcgcg gccggcacag
     4621 tccccatcta cgtaccctcg tgaactttcc catgccgttt cagcctgctg ggcgaggcaa
     4681 agtgcttccc gcatccctcg tgggtacacc taggacaata gcaggcaaag catcagccag
     4741 gccctttcca tggacaattc taagattccc tcttctccca caccaaagcc catgtcttcc
     4801 cacaccacac ctgtcatcca aaacaaagag taagtacagt cccgaatatg gacatagtgg
     4861 cttggccagc ttgggagcaa aacaagccat cctgagaaaa agtcacatct tttgtttgtg
     4921 ttatcaaact aaaagttctc tgaattctgg aaatgtggct acagaagcga catcaagagg
     4981 ctgtctgact cggtttgcac tggaccagac acagagctgt tgtctggggt ctgcccaata
     5041 tgctgcatac ggtcagatga gaacacagca cagaggctgg ggaccagcac cagaaacaca
     5101 cgagaatgta cagcagtcgg tgcagagctc caggcactgg cagcacaaac aggccacagg
     5161 gctgctggta agacctcggt cactgtagct gcaccagtgc aaagagcagc ctatgggagc
     5221 caggggcaag gccccatcac ccggactgtg cagggagaca ctgccctagg agggaggcag
     5281 gcaggtaacc tggcacccct gcccacctcc tctttgtccc tcttgagtac tcaggaatcc
     5341 ctcaaaacaa gcagggcctc attactcctg ttctatagta agaaaaaaaa aagccctcat
     5401 gacacagagc cttcctcggg atcctgatct tagcgttcta ctctgaatcc cagagcgctg
     5461 gcaaagcacc aacctgaaga gcggctcgct ggtgtgctgg cactgatggg ttctcagctg
     5521 ctggtgcttc ttaaaggcct tcttgcaacc ctcataactg cactgtttaa aaaaaataaa
     5581 tgcacggttt agtacaggca ggggatcatc agttgctgaa gtcctttaaa aggcaggaga
     5641 gattgctcaa tggttaggga cacttgcttc tctggtccca ggtttggact gcagcaccca
     5701 cgtggtgcct cacaaccatc tgactccagt tccacaggac ctcacaccct cttctgacct
     5761 ccacaggcac cgagcacgca tatggtatat agatacatat gcaggcaaaa cgcttgcaca
     5821 taaaaccttt tttaactaaa caataaagtc tctctggatc tgaccggtca tcatcaccat
     5881 caccattgag tttggccgct ggccgcaata aaatatcttt attttcatta catctgtgtg
     5941 ttggtttttt gtgtggaatt ccgttacata acttacggta aatggcccgc ctggctgacc
     6001 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     6061 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     6121 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     6181 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6241 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     6301 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     6361 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     6421 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     6481 ccgtcagatc cgctagcatg tccgtcctga cgccgctgct gctgcggggc ttgacaggct
     6541 cggcccggcg gctcccagtg ccgcgcgcca agatccattc gttgggggat ctgtccgtcc
     6601 tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca gtgccgcgcg
     6661 ccaagatcca ttcgttgggg gatccaccgg tcgccaccat ggtgagcaag ggcgaggaga
     6721 ccacaatggg cgtaatcaag cccgacatga agatcaagct gaagatggag ggcaacgtga
     6781 atggccacgc cttcgtgatc gagggcgagg gcgagggcaa gccctacgac ggcaccaaca
     6841 ccatcaacct ggaggtgaag gagggagccc ccctgccctt ctcctacgac attctgacca
     6901 ccgcgttcgc ctacggcaac agggccttca ccaagtaccc cgacgacatc cccaactact
     6961 tcaagcagtc cttccccgag ggctactctt gggagcgcac catgaccttc gaggacaagg
     7021 gcatcgtgaa ggtgaagtcc gacatctcca tggaggagga ctccttcatc tacgagatac
     7081 acctcaaggg cgagaacttc ccccccaacg gccccgtgat gcagaagaag accaccggct
     7141 gggacgcctc caccgagagg atgtacgtgc gcgacggcgt gctgaagggc gacgtcaagc
     7201 acaagctgct gctggagggc ggcggccacc accgcgttga cttcaagacc atctacaggg
     7261 ccaagaaggc ggtgaagctg cccgactatc actttgtgga ccaccgcatc gagatcctga
     7321 accacgacaa ggactacaac aaggtgaccg tttacgagag cgccgtggcc cgcaactcca
     7381 ccgacggcat ggacgagctg tacaagtaac tcgagtctag agtcggggcg gccggccgct
     7441 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     7501 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     7561 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     7621 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga
     7681 tccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     7741 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     7801 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     7861 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     7921 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     7981 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     8041 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     8101 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     8161 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     8221 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     8281 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     8341 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     8401 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     8461 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     8521 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     8581 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     8641 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     8701 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     8761 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     8821 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     8881 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     8941 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     9001 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     9061 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     9121 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     9181 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     9241 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     9301 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     9361 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     9421 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     9481 ttttgctcac atggctcgac
//
