LOCUS       sequence-421714-        7572 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 7572)
  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 7572)
  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 43-684
FEATURES             Location/Qualifiers
     source          1..7572
                     /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(2518..2811)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(2818..2835)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2845..2862)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2882..2904
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2919..2943
                     /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(2943..2962)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2944..2962)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3046..3065)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3271..3348
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3349..4431
                     /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(3368..3387)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5240..5431
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5425..5447)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5533..5672
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5588..5607
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5687..5704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5858..6446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5938..5957)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6557..6576)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6568..7383
                     /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             6568..7383
                     /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(6641..6660)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(7475..7572,1..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 aggcagggcg gtacgaagaa tacgagaaag attgaagcct ccgttcaggg cctccagggg
     1621 acggggttgc tggtctcctg gtcgcaccaa gaagcatcgt ttggggttca gttttcgtgc
     1681 aaacaacttg gcggcttccc aggaccgaag gtcatccccc aaccagaata caatccgccg
     1741 gaactgttcc aggtaaggga gtaaggcagg gggtaagcag gtcgttcctc ggggtagagt
     1801 aagggtaggc agccccgtgg actggttcaa ggccaggctg tcaagctcac gactcgtcag
     1861 taccacctca gcatctcgac gactaatcag tggtaatcca aacagattgt ggtaggcgct
     1921 gggtcgggga atagtggttt cctcgtagct cactccatcc ccctggcatt tagcctctag
     1981 gagcttcagg cctcgtaatc ctgagccccc aggggagaac caagggaaga caagactgcg
     2041 agcaggtcgc agatatcgca cactgaaacg cttgagtgtg tcatctgtaa ccttggtaag
     2101 gccaaacatt gtatcagcca gctgaacctc ctcctgatca ggcagctccc agagaggtat
     2161 tgctcggttc cagatcctcc ggacctcctc gctgtcctca aattctggtg ccttgctaag
     2221 cagaaacccc tccctggccc catcccctcg cccctccacg ctggcctgga agtcttccca
     2281 gctcccttct gctaggctgg tcatgcagag aaagtggcct gtggtcttgt caatgaagag
     2341 gctgaaggaa gtggtaacac cagtctggcc tttgagctgt gaagactctg caaaggggct
     2401 cagtgcccgc aggcaactgt gaccatcctg gaaggggatc ccatgccccc gcaaatactg
     2461 gcggatttca gttgcagtta caggcaacac tggcatatcc aaggcttgga gagtctcacc
     2521 accaatctgt tctctgtgag cctcaataat atcgttatcc tccatgtcca aatcttcagg
     2581 ggtctgatca gcttgaattc taataccgtc gtacaagaat cttaaggagt ccatttcctt
     2641 accctgtctt ttagcgaacg cttccatcag ccttcttaaa ggagtggtct ttttgatctt
     2701 gaagaagatc tctgaagatc catcggacac ctttaaattg atgtgagtct caggcttgac
     2761 ttctggcttg acctctggct tagcttcttg attgacttct gagtccgaca tgctagcgct
     2821 gccgcgcggc accaggccgc tgccgtgatg atgatgatga tggctgctgc ccatatgtat
     2881 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
     2941 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     3001 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     3061 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     3121 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     3181 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     3241 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     3301 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     3361 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     3421 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     3481 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     3541 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     3601 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     3661 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     3721 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     3781 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     3841 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     3901 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     3961 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     4021 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     4081 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     4141 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     4201 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     4261 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     4321 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     4381 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     4441 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     4501 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     4561 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt
     4621 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     4681 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     4741 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     4801 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     4861 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     4921 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     4981 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     5041 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     5101 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     5161 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     5221 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     5281 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     5341 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     5401 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     5461 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5521 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     5581 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     5641 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     5701 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5761 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5821 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5881 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5941 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6001 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6061 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6121 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6181 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6241 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6301 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6361 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6421 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6481 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     6541 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     6601 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     6661 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     6721 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     6781 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     6841 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     6901 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     6961 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     7021 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     7081 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     7141 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     7201 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     7261 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     7321 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     7381 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7441 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     7501 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     7561 aaaatccctt at
//
