LOCUS       sequence-413735-        6588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TWNK protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6588)
  AUTHORS   Johnson LC, Singh A, Patel SS
  TITLE     The N-terminal domain of human mitochondrial helicase Twinkle has 
            DNA-binding activity crucial for supporting processive DNA synthesis
            by polymerase gamma.
  JOURNAL   J Biol Chem. 2023 Jan;299(1):102797. doi: 10.1016/j.jbc.2022.102797.
            Epub 2022 Dec 14.
  PUBMED    36528058
REFERENCE   2  (bases 1 to 6588)
  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: "J Biol 
            Chem."; date: "2023-01"; volume: "299(1):102797. doi"; pages: " 
            10.1016/j.jbc.2022.102797. Epub 2022 Dec 14"
COMMENT     Sequence Label: SUMO-Twinkle 43-372
FEATURES             Location/Qualifiers
     source          1..6588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..39)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     230..249
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(373..420)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     416..434
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(487..504)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1507..1800)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(1813..1830)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1840..1857)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1876..1898
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1913..1937
                     /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     complement(1937..1956)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1938..1956)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2040..2059)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2265..2342
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2343..3425
                     /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     complement(2362..2381)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4234..4425
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4419..4441)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4527..4666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4582..4601
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4681..4698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4852..5440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4932..4951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5551..5570)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5562..6377
                     /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             5562..6377
                     /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     complement(5635..5654)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6469..6588,1..336))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
       61 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      121 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      181 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      241 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      301 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt
      361 tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag
      421 ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg
      481 atctcagtgg tggtggtggt ggtgctcgag ttactgccgg aaagatacga tggacttgtg
      541 ccaggcaggc agggcggtac gaagaatacg agaaagattg aagcctccgt tcagggcctc
      601 caggggacgg ggttgctggt ctcctggtcg caccaagaag catcgtttgg ggttcagttt
      661 tcgtgcaaac aacttggcgg cttcccagga ccgaaggtca tcccccaacc agaatacaat
      721 ccgccggaac tgttccaggt aagggagtaa ggcagggggt aagcaggtcg ttcctcgggg
      781 tagagtaagg gtaggcagcc ccgtggactg gttcaaggcc aggctgtcaa gctcacgact
      841 cgtcagtacc acctcagcat ctcgacgact aatcagtggt aatccaaaca gattgtggta
      901 ggcgctgggt cggggaatag tggtttcctc gtagctcact ccatccccct ggcatttagc
      961 ctctaggagc ttcaggcctc gtaatcctga gcccccaggg gagaaccaag ggaagacaag
     1021 actgcgagca ggtcgcagat atcgcacact gaaacgcttg agtgtgtcat ctgtaacctt
     1081 ggtaaggcca aacattgtat cagccagctg aacctcctcc tgatcaggca gctcccagag
     1141 aggtattgct cggttccaga tcctccggac ctcctcgctg tcctcaaatt ctggtgcctt
     1201 gctaagcaga aacccctccc tggccccatc ccctcgcccc tccacgctgg cctggaagtc
     1261 ttcccagctc ccttctgcta ggctggtcat gcagagaaag tggcctgtgg tcttgtcaat
     1321 gaagaggctg aaggaagtgg taacaccagt ctggcctttg agctgtgaag actctgcaaa
     1381 ggggctcagt gcccgcaggc aactgtgacc atcctggaag gggatcccat gcccccgcaa
     1441 atactggcgg atttcagttg cagttacagg caacactggc atatccaagg cttggagagt
     1501 ctcggatcca ccaatctgtt ctctgtgagc ctcaataata tcgttatcct ccatgtccaa
     1561 atcttcaggg gtctgatcag cttgaattct aataccgtcg tacaagaatc ttaaggagtc
     1621 catttcctta ccctgtcttt tagcgaacgc ttccatcagc cttcttaaag gagtggtctt
     1681 tttgatcttg aagaagatct ctgaagatcc atcggacacc tttaaattga tgtgagtctc
     1741 aggcttgact tctggcttga cctctggctt agcttcttga ttgacttctg agtccgacat
     1801 gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct
     1861 gctgcccatg gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt
     1921 tatccgctca caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat
     1981 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     2041 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     2101 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     2161 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     2221 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     2281 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     2341 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     2401 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     2461 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     2521 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     2581 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     2641 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     2701 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     2761 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     2821 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     2881 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     2941 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     3001 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     3061 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     3121 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     3181 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     3241 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     3301 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     3361 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     3421 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     3481 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     3541 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     3601 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     3661 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     3721 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
     3781 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     3841 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     3901 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     3961 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     4021 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     4081 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     4141 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     4201 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     4261 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     4321 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     4381 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4441 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4501 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     4561 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg
     4621 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     4681 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4741 aatggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4801 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4861 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4921 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4981 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5041 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5101 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5161 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5221 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5281 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5341 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5401 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5461 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa
     5521 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     5581 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     5641 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     5701 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     5761 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     5821 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     5881 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     5941 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     6001 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     6061 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     6121 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     6181 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     6241 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     6301 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     6361 tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa
     6421 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     6481 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6541 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgaga
//
