LOCUS       sequence-358111-        4370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression construct encoding 3xFLAG and mito-mTFP1 from 
            separate promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4370)
  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 4370)
  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-3xFLAG-CMVp-mito-mTFP1
FEATURES             Location/Qualifiers
     source          1..4370
                     /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             693..758
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    777..825
                     /note="synthetic polyadenylation signal"
     enhancer        832..1135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1136..1339
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1290..1310
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1368..1442
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1464..1535
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      1563..1572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1569..2279
                     /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    2325..2446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2336..2355)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2390..2409
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2579..2683
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2684..2752,2753..3544)
                     /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(2902..2921)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3715..4303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4204..4223
                     /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 taggctagca tggactataa ggaccacgac ggagactaca
      721 aggatcatga tattgattac aaagacgatg acgataagta agcggccgct ggccgcaata
      781 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtggaatt ccgttacata
      841 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      901 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      961 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1021 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1081 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1141 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1201 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1261 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1321 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcatg tccgtcctga
     1381 cgccgctgct gctgcggggc ttgacaggct cggcccggcg gctcccagtg ccgcgcgcca
     1441 agatccattc gttgggggat ctgtccgtcc tgacgccgct gctgctgcgg ggcttgacag
     1501 gctcggcccg gcggctccca gtgccgcgcg ccaagatcca ttcgttgggg gatccaccgg
     1561 tcgccaccat ggtgagcaag ggcgaggaga ccacaatggg cgtaatcaag cccgacatga
     1621 agatcaagct gaagatggag ggcaacgtga atggccacgc cttcgtgatc gagggcgagg
     1681 gcgagggcaa gccctacgac ggcaccaaca ccatcaacct ggaggtgaag gagggagccc
     1741 ccctgccctt ctcctacgac attctgacca ccgcgttcgc ctacggcaac agggccttca
     1801 ccaagtaccc cgacgacatc cccaactact tcaagcagtc cttccccgag ggctactctt
     1861 gggagcgcac catgaccttc gaggacaagg gcatcgtgaa ggtgaagtcc gacatctcca
     1921 tggaggagga ctccttcatc tacgagatac acctcaaggg cgagaacttc ccccccaacg
     1981 gccccgtgat gcagaagaag accaccggct gggacgcctc caccgagagg atgtacgtgc
     2041 gcgacggcgt gctgaagggc gacgtcaagc acaagctgct gctggagggc ggcggccacc
     2101 accgcgttga cttcaagacc atctacaggg ccaagaaggc ggtgaagctg cccgactatc
     2161 actttgtgga ccaccgcatc gagatcctga accacgacaa ggactacaac aaggtgaccg
     2221 tttacgagag cgccgtggcc cgcaactcca ccgacggcat ggacgagctg tacaagtaac
     2281 tcgagtctag agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga
     2341 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     2401 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2461 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     2521 cctctacaaa tgtggtaaaa tcgataagga tccaggtggc acttttcggg gaaatgtgcg
     2581 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2641 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2701 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2761 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2821 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2881 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2941 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3001 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3061 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3121 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3181 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3241 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3301 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3361 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3421 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3481 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3541 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3601 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3661 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3721 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3781 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3841 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3901 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3961 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4021 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4081 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4141 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4201 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4261 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4321 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atggctcgac
//
