LOCUS       sequence-382263-        4450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of AtTIM21-mCherry in plant cell 
            (Mitochondria).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4450)
  AUTHORS   Ichikawa S, Kato S, Fujii Y, Ishikawa K, Numata K, Kodama Y
  TITLE     Organellar Glue: A Molecular Tool to Artificially Control 
            Chloroplast-Chloroplast Interactions.
  JOURNAL   ACS Synth Biol. 2022 Oct 21;11(10):3190-3197. doi: 
            10.1021/acssynbio.2c00367. Epub 2022 Sep 30.
  PUBMED    36178266
REFERENCE   2  (bases 1 to 4450)
  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"; doi: 
            "10.1021/acssynbio.2c00367"; journalName: "ACS Synth Biol"; date: 
            "2022-10-21- 21"; volume: "11"; issue: "10"; pages: "3190-3197"
COMMENT     Sequence Label: pGWT35S-AtTIM21(1-50aa)-mCherry
FEATURES             Location/Qualifiers
     source          1..4450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     405..424
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        405..423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        500..844
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     787..807
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     misc_feature    864..919
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     protein_bind    963..987
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1140..1850
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1285..1303)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1553..1572
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1855..1879)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      1924..2174
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        complement(2239..2257)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2240..2257)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2275..2291)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2275..2291)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2288..2310)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2299..2315
                     /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)."
     promoter        complement(join(2323..2329,2330..2347,2348..2353))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2368..2389
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2506..2523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2677..3265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2757..2776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3423..4238)
                     /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     4146..4165
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4230..4249
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4423..4441
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaatattac tacaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgagct agtgtaatac gactcactat
      421 agggcgcggc cgcagaattc gagctcggta cccgggatct cgaggccaga tctcccgggc
      481 tcagaagacc gagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg
      541 gattccattg cccagctatc tgtcacttat cgaaaggaca gtagaaaagg aaggtggctc
      601 ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctaccgacag
      661 tggtcccaaa gatggacccc cacccacgag gaacatcgtg gaaaaagaag acgttccaac
      721 cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca
      781 atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag
      841 gacaggcttc ttgagatcct tcaacaatta ccaacaacaa caaacaacaa acaacattac
      901 aattactatt tacaattaca gaattcgagc tcggtaccaa gcttggaggc tcaggaggct
      961 ccacaagttt gtacaaaaaa gcaggctcca tgatgatgat gaatctttta aggagatcag
     1021 cgatagccat cggccgccaa tcaaaatcga aactggcgtc gttttcctcc gctacacaac
     1081 cttgttccgg aatcccaaaa tcgtccaaga gggtttttag caactcattt ctttcaaaga
     1141 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1201 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1261 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     1321 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     1381 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1441 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1501 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     1561 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     1621 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     1681 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     1741 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     1801 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtag tgagacccag
     1861 ctttcttgta caaagtggtt ggaggctcag gaggctcccc atgggctagc actagtatct
     1921 agaatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg
     1981 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat
     2041 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat
     2101 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat
     2161 ctatgttact agatgcggcc gcaattgtgg atccgctcta gagtcgacct gcaggcatgc
     2221 aagcttgcgg ccgcgtattc tatagtgtca cctaaatagc atggcgtaat catggtcata
     2281 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2341 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2401 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2461 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2521 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2581 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2641 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2701 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2761 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2821 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2881 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2941 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3001 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3061 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3121 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3181 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3241 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3301 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3361 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgtttt
     3421 aattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3481 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3541 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3601 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3661 actgaatccg gtgagaatgg caatagctta tgcatttctt tccagacttg ttcaacaggc
     3721 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3781 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3841 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3901 tattcttcta atacctggaa tgctgttttc cctgggatcg cagaggtgag taaccatgca
     3961 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4021 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4081 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     4141 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4201 ggccttgagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4261 atgtaagcag acataagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4321 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4381 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4441 ccctttcgtc
//
