LOCUS       sequence-329812-        5619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TFAM HMGB+C-tatil mutant in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5619)
  AUTHORS   Feric M, Demarest TG, Tian J, Croteau DL, Bohr VA, Misteli T
  TITLE     Self-assembly of multi-component mitochondrial nucleoids via phase 
            separation.
  JOURNAL   EMBO J. 2021 Mar 15;40(6):e107165. doi: 10.15252/embj.2020107165. 
            Epub 2021 Feb 23.
  PUBMED    33619770
REFERENCE   2  (bases 1 to 5619)
  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: "EMBO J."; 
            date: "2021-03-15"; volume: "40(6):e107165. doi"; pages: " 
            10.15252/embj.2020107165. Epub 2021 Feb 23"
COMMENT     Sequence Label: Human_TFAM_HMGB_ctail_pET28
FEATURES             Location/Qualifiers
     source          1..5619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             129..161
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             465..482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(535..553)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      549..596
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      633..1088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(720..739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     930..951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1180..1995)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1180..1995)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1903..1922
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1987..2006
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2117..2705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2606..2625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2859..2876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2891..3030
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2956..2975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3116..3138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3132..3323)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4132..5214)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5176..5195
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5215..5292)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5498..5517
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5601..5619,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5601..5619
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat ggctagcatg actggtggac agcaaatggg tcgcggatcc atgggaaaac
      181 caaaaagacc tcgttcagct tataacgttt atgtagctga aagattccaa gaagctaagg
      241 gtgattcacc gcaggaaaag ctgaagactg taaaggaaaa ctggaaaaat ctgtctgact
      301 ctgaaaagga attatatatt cagcatgcta aagaggacga aactcgttat cataatgaaa
      361 tgaagtcttg ggaagaacaa atgattgaag ttggacgaaa ggatcttcta cgtcgcacaa
      421 taaagaaaca acgaaaatat ggtgctgagg agtgttaact cgagcaccac caccaccacc
      481 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
      541 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
      601 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      661 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      721 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      781 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      841 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      901 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      961 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1021 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1081 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1141 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1201 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1261 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1321 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1381 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1441 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1501 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1561 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1621 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1681 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1741 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1801 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1861 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     1921 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     1981 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2041 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2821 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2881 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2941 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3001 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3061 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3121 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3181 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3241 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3301 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3361 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3421 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3481 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3541 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3601 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3661 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3721 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3781 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3841 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3901 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3961 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4021 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4081 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4141 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4201 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4261 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4321 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4381 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     4441 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4501 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4561 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4621 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4681 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4741 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4801 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4861 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4921 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4981 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5041 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5101 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5161 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5221 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5281 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5341 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5401 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5461 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5521 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     5581 agatctcgat cccgcgaaat taatacgact cactatagg
//
