LOCUS       sequence-361290-        4456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of TIM21(1–50)-Citrine in plant cell 
            (Mitochondria).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4456)
  AUTHORS   Osaki Y, Kodama Y
  TITLE     Particle bombardment and subcellular protein localization analysis 
            in the aquatic plant Egeria densa.
  JOURNAL   PeerJ. 2017 Sep 7;5:e3779. doi: 10.7717/peerj.3779. eCollection 
            2017.
  PUBMED    28894649
REFERENCE   2  (bases 1 to 4456)
  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: "PeerJ."; 
            date: "2017-09-7"; volume: "5:e3779. doi"; pages: " 
            10.7717/peerj.3779. eCollection 2017"
COMMENT     Sequence Label: pGWT35S-TIM21(1–50)-Citrine
FEATURES             Location/Qualifiers
     source          1..4456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     160..179
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     419..440
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(638..662)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    706..761
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     promoter        complement(781..1125)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(818..838)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     primer_bind     complement(1201..1220)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1202..1220)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1230..1247)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1230..1246)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1239..1261)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1455..1474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1574..1596
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1634..1652)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1826..1845)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1837..2652
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTSAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1910..1929)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2810..3398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3299..3318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3552..3569
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3686..3707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3722..3727,3728..3745,3746..3752)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3760..3776
                     /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)."
     primer_bind     3765..3787
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3784..3800
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3784..3800
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3818..3836
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3818..3835
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(3901..4151)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4196..4220
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4225..4456,1..479,480..482,483..485))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4267..4288)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
       61 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
      121 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
      181 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
      241 tgctgcttca tgtggtcggg gtagcgggcg aagcacatca ggccgtagcc gaaggtggtc
      301 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
      361 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
      421 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
      481 accatctttg aaagaaatga gttgctaaaa accctcttgg acgattttgg gattccggaa
      541 caaggttgtg tagcggagga aaacgacgcc agtttcgatt ttgattggcg gccgatggct
      601 atcgctgatc tccttaaaag attcatcatc atcatggagc ctgctttttt gtacaaactt
      661 gtggagcctc ctgagcctcc aagcttggta ccgagctcga attctgtaat tgtaaatagt
      721 aattgtaatg ttgtttgttg tttgttgttg ttggtaattg ttgaaggatc tcaagaagcc
      781 tgtcctctcc aaatgaaatg aacttcctta tatagaggaa gggtcttgcg aaggatagtg
      841 ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga
      901 cgtggttgga acgtcttctt tttccacgat gttcctcgtg ggtgggggtc catctttggg
      961 accactgtcg gtagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt
     1021 gtaggagcca ccttcctttt ctactgtcct ttcgataagt gacagatagc tgggcaatgg
     1081 aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctcggtctt
     1141 ctgagcccgg gagatctggc ctcgagatcc cgggtaccga gctcgaattc tgcggccgcg
     1201 ccctatagtg agtcgtatta cactagctca ctggccgtcg ttttacaacg tcgtgactgg
     1261 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     1321 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     1381 gaatggcgcc tgtagtaata ttttctcctt acgcatctgt gcggtatttc acaccgcata
     1441 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     1501 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     1561 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     1621 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1681 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1741 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1801 atgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     1861 tcttgctcaa ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1921 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1981 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     2041 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     2101 cgtactcctg atgatgcatg gttactcacc tctgcgatcc cagggaaaac agcattccag
     2161 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     2221 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     2281 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2341 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct attgccattc
     2401 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2461 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2521 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2581 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2641 gagtttttct aattaaaaca tatatacttt agattgattt aaaacttcat ttttaattta
     2701 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2761 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2821 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2881 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2941 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3001 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3061 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3121 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3181 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3241 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3301 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3361 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3421 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3481 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3541 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     3601 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3661 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     3721 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     3781 acacaggaaa cagctatgac catgattacg ccatgctatt taggtgacac tatagaatac
     3841 gcggccgcaa gcttgcatgc ctgcaggtcg actctagagc ggatccacaa ttgcggccgc
     3901 atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc gctatatttt
     3961 gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa cccatctcat
     4021 aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca acagaaatta
     4081 tatgataatc atcgcaagac cggcaacagg attcaatctt aagaaacttt attgccaaat
     4141 gtttgaacga ttctagatac tagtgctagc ccatggggag cctcctgagc ctccaaccac
     4201 tttgtacaag aaagctgggt ctcacttgta cagctcgtcc atgccgagag tgatcccggc
     4261 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     4321 ggcggactgg tagctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     4381 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     4441 cttgaagttc accttg
//
