LOCUS       sequence-358102-        5201 bp    DNA     circular SYN 15-APR-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 5201)
  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 5201)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-mtIF3(SR)-3xFLAG-CMVp-mito-mTFP1
FEATURES             Location/Qualifiers
     source          1..5201
                     /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             1524..1589
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1608..1656
                     /note="synthetic polyadenylation signal"
     enhancer        1663..1966
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1967..2170
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2121..2141
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2199..2273
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2295..2366
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      2394..2403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2400..3110
                     /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    3156..3277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3167..3186)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3221..3240
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3410..3514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3515..3583,3584..4375)
                     /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(3733..3752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4546..5134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5035..5054
                     /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 tggctgttct tctgatgagg ttgatgctac
      721 aaactacaaa gcttgaccat aacttaattg gaagatgttt gcaaagacac gctgtgaagc
      781 cagatccagc acagctgtcc ctgtctgcgt ccaccccaaa gctgttgtac ctgacttctg
      841 caaaaggttt tagtactgct ggagacccac agggtgagag gaaacagaaa agaagggatg
      901 ctttctctaa caccggccgg aagattagtg agcggattat ccgcgtcctg gatgagaagg
      961 gcatggacct ggggatgatg catcgagcag atgtgatcag actcatgaac aagcaggacc
     1021 tgaggctggt gcagaggaac accagctcag agccaccgga gtaccagctt atgaccggag
     1081 agcagatcca ccaggagcgg ctaaagctcc gggaacagga aaaggccaaa cccaaaactg
     1141 gaccaactat gacgaaagag ctcgttttct cttcaaatat tggacagcac gatttagaca
     1201 ccaagagtaa acagatccag cagtggatag agaagaaata tcatgtacag gtgaccatta
     1261 agaggaggaa agatgcggag cagtcagagg aggaaacgga ggagatattt aaccagattc
     1321 tccagactat gcctgacata gcaaccttct caagcagacc aaaagccatc agaggaggga
     1381 ctgcttccat gtgtgttttt cgtcacttga gcaagaaaga agaaaaggcg tacagagaat
     1441 cccaggagag ccagagaaga gacactttga gcaaagacga cgacggaaac agcaaagagt
     1501 cggatgttgt gtgtcagacc ggtgactata aggaccacga cggagactac aaggatcatg
     1561 atattgatta caaagacgat gacgataagt aagcggccgc tggccgcaat aaaatatctt
     1621 tattttcatt acatctgtgt gttggttttt tgtgtggaat tccgttacat aacttacggt
     1681 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1741 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1801 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1861 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1921 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     1981 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2041 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2101 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2161 aagcagagct ggtttagtga accgtcagat ccgctagcat gtccgtcctg acgccgctgc
     2221 tgctgcgggg cttgacaggc tcggcccggc ggctcccagt gccgcgcgcc aagatccatt
     2281 cgttggggga tctgtccgtc ctgacgccgc tgctgctgcg gggcttgaca ggctcggccc
     2341 ggcggctccc agtgccgcgc gccaagatcc attcgttggg ggatccaccg gtcgccacca
     2401 tggtgagcaa gggcgaggag accacaatgg gcgtaatcaa gcccgacatg aagatcaagc
     2461 tgaagatgga gggcaacgtg aatggccacg ccttcgtgat cgagggcgag ggcgagggca
     2521 agccctacga cggcaccaac accatcaacc tggaggtgaa ggagggagcc cccctgccct
     2581 tctcctacga cattctgacc accgcgttcg cctacggcaa cagggccttc accaagtacc
     2641 ccgacgacat ccccaactac ttcaagcagt ccttccccga gggctactct tgggagcgca
     2701 ccatgacctt cgaggacaag ggcatcgtga aggtgaagtc cgacatctcc atggaggagg
     2761 actccttcat ctacgagata cacctcaagg gcgagaactt cccccccaac ggccccgtga
     2821 tgcagaagaa gaccaccggc tgggacgcct ccaccgagag gatgtacgtg cgcgacggcg
     2881 tgctgaaggg cgacgtcaag cacaagctgc tgctggaggg cggcggccac caccgcgttg
     2941 acttcaagac catctacagg gccaagaagg cggtgaagct gcccgactat cactttgtgg
     3001 accaccgcat cgagatcctg aaccacgaca aggactacaa caaggtgacc gtttacgaga
     3061 gcgccgtggc ccgcaactcc accgacggca tggacgagct gtacaagtaa ctcgagtcta
     3121 gagtcggggc ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     3181 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3241 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3301 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     3361 atgtggtaaa atcgataagg atccaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4681 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5161 gttcctggcc ttttgctggc cttttgctca catggctcga c
//
