LOCUS       sequence-413731-        6624 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 6624)
  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 6624)
  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 360-684
FEATURES             Location/Qualifiers
     source          1..6624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..61)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     252..271
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(395..442)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     438..456
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(509..526)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1570..1863)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(1870..1887)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1897..1914)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1934..1956
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1971..1995
                     /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(1995..2014)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1996..2014)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2098..2117)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2323..2400
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2401..3483
                     /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(2420..2439)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4292..4483
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4477..4499)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4585..4724
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4640..4659
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4739..4756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4910..5498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4990..5009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5609..5628)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5620..6435
                     /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             5620..6435
                     /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(5693..5712)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6527..6624,1..358))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
       61 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
      121 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
      181 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
      241 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
      301 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
      361 cattcgccaa tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag
      421 aggccccaag gggttatgct agttattgct cagcggtggc agcagccaac tcagcttcct
      481 ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg actaagttgg
      541 cagcatcacc cgacgcactt tgcgccgaat aaatacctaa gcttgtcttt actttgaacg
      601 cttggaggtg tctggctggt cgggagtggg ggcctggcct gagcaaatct cagagttctg
      661 tgtcgtagcc ccctttttgc cagaagaggg ctttttggcc actggtccag tgtcatcctt
      721 gatcttcttg agccgggcct tgttctttgg tggaatggag aaggtgaggg agttcttgtt
      781 gaactcaagc gggaagacac ctacatctcc atcaaagcgg ttcttggaca cctgcagata
      841 ccgtttccct ggcccggtta ccagcttcct gtcctgcagg atcagaacat tgtctgcttc
      901 ctggcttgct ttggctgagc caaaaatgga cgctgtctgc agttccttgt catcatcctc
      961 tttccggggg tgaatgacca gtgtcacatg gcagttattg tctgttgcaa acttccgaaa
     1021 gaccccgatg atgtagtctt gagctgcgat cctgtctgtg gacagctgct cgtgacccat
     1081 catgaactgc aggttgtcga tgatcacatg acaaatgtca tagacgtaga ctgcatgttg
     1141 cattgtatct attacagtcc tgatgctttg ctgtccatgg aaagtcatga aatagagggg
     1201 caggtcctca aagcggtcag cccagtgatc atatttgtcc agttgatctt ccagccgccc
     1261 ctcggcaaac tgtgtcagca tgacccgggc tagtctcaca ttgctgatct cgaagctacc
     1321 ccacagtgtg ttcaccccct gggaacacaa atccagggca tactcactga tgaatgtcgt
     1381 ctttccactg cctgttggcc ctgtgaagac cgtcagctcg ccctttcgat gtcccttcaa
     1441 gatacgattg aggtctggaa agcggctcca gcggaggcca gctgcttgct ccacatttga
     1501 cagttctcct agcacctcct cccgaagctg ccggaaagat acgatggact tgtgccaggc
     1561 aggcagggat ccaccaatct gttctctgtg agcctcaata atatcgttat cctccatgtc
     1621 caaatcttca ggggtctgat cagcttgaat tctaataccg tcgtacaaga atcttaagga
     1681 gtccatttcc ttaccctgtc ttttagcgaa cgcttccatc agccttctta aaggagtggt
     1741 ctttttgatc ttgaagaaga tctctgaaga tccatcggac acctttaaat tgatgtgagt
     1801 ctcaggcttg acttctggct tgacctctgg cttagcttct tgattgactt ctgagtccga
     1861 catgctagcg ctgccgcgcg gcaccaggcc gctgccgtga tgatgatgat gatggctgct
     1921 gcccatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     1981 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     2041 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     2101 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     2161 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     2221 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     2281 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     2341 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     2401 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     2461 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     2521 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     2581 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     2641 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     2701 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     2761 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     2821 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     2881 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     2941 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     3001 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     3061 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     3121 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     3181 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     3241 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     3301 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     3361 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     3421 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3481 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     3541 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     3601 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     3661 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     3721 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     3781 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     3841 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     3901 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     3961 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     4021 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     4081 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     4141 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     4201 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     4261 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     4321 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     4381 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     4441 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     4501 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     4561 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     4621 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     4681 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4741 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4801 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4861 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4921 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4981 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5041 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5101 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5161 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5221 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5281 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5341 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5401 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5461 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5521 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     5581 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     5641 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5701 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     5761 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5821 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5881 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     5941 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     6001 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     6061 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     6121 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     6181 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     6241 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     6301 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     6361 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     6421 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     6481 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     6541 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6601 gccgaaatcg gcaaaatccc ttat
//
