LOCUS       sequence-157191-        6589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of catalytically dead murine TDG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6589)
  AUTHORS   Weber AR, Krawczyk C, Robertson AB, Kusnierczyk A, Vagbo CB, 
            Schuermann D, Klungland A, Schar P
  TITLE     Biochemical reconstitution of TET1-TDG-BER-dependent active DNA 
            demethylation reveals a highly coordinated mechanism.
  JOURNAL   Nat Commun. 2016 Mar 2;7:10806. doi: 10.1038/ncomms10806.
  PUBMED    26932196
REFERENCE   2  (bases 1 to 6589)
  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: "Nat 
            Commun."; date: "2016-03-2"; volume: "7:10806. doi"; pages: " 
            10.1038/ncomms10806"
COMMENT     Sequence Label: pET28-mTDGa.1
FEATURES             Location/Qualifiers
     source          1..6589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..78)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     284..303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     387..406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        387..405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    406..430
                     /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             445..467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             486..503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             513..530
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1836..1853
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1906..1924)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1920..1967
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2004..2459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2091..2110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2301..2322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2552..3367)
                     /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(2552..3367)
                     /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     3275..3294
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3359..3378
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3489..4077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3978..3997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4231..4248
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4263..4405
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4331..4350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4491..4513
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4507..4698)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5507..6589)
                     /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     6551..6570
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
       61 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
      121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      181 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      241 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      301 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      361 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
      421 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
      481 gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggcta
      541 gcatggacgc agaggccgcg cgcagctatt ctctggagca agttcaagct ttgtattcat
      601 ttccatttca acaaatgatg gcagaagttc ctaacatggc agtcacgact ggacagcagg
      661 tgccagcagt agctcctaac atggcaaccg tgacagaaca gcaggtgccc gcagacgctc
      721 ctgtccagga acctgcacca gaagctccaa agagaaggaa aaggaaaccc agagcagcag
      781 agccccagga accagtggag cccaaaaaac ctgctacgtc gaagaaatcc ggcaagtcta
      841 caaaatcaaa ggaaaagcag gagaaaatca cagacgcgtt taaagtgaaa aggaaagtgg
      901 accgcttcaa cggcgtctct gaagctgagc ttctgaccaa gactcttcct gacattttga
      961 ccttcaatct ggatattgtg atcattggca ttgccccggg attaatggct gcttacaaag
     1021 gacatcacta ccctgggcct ggaaatcact tctggaagtg tctgttcatg tcggggctga
     1081 gtgaggtgca gctgaatcac atggatgacc acaccttacc cggcaagtac ggcatcggat
     1141 tcaccaacat ggtggaacgg acgacgccgg gcagcaagga tctgtctagt aaagagttcc
     1201 gggaaggagg gcgcatcctg gtgcagaaac tgcagaaata tcagccacga atagcggtgt
     1261 ttaatggaaa atgtatttat gaaattttca gtaaagaagt ttttggagta aaggttaaga
     1321 acttggaatt tgggcttcaa ccccacaaga tcccagacac agaaactctg tgctacgtca
     1381 tgccgtcgtc cagcgccaga tgtgctcagt ttccccgggc ccaggacaaa gttcattact
     1441 acattaagct gaaggacttg agagaccagc tgaaaggcat tgaacgcaac accgacgttc
     1501 aggaagtgca gtatacattt gacctgcagc ttgcgcaaga ggacgcaaag aagatggctg
     1561 ttaaggaaga aaagtatgat ccaggctatg aggcagctta cggcggcgcc tatggggaaa
     1621 acccatgtaa tggggaacct tgtggcattg cttcaaatgg gctaacagcc cacagtgcgg
     1681 agccgagagg agaagcgacc cccggcgatg ttccaaatgg gcagtggatg gcacagtcgt
     1741 ttgcagagca gatcccttct tttaataatt gtgggacccg agagcaggaa gaagagagcc
     1801 acgcttaggt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     1861 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1921 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1981 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2041 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2101 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2161 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2221 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2281 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2341 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2401 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2461 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2521 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2581 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2641 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2701 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2761 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2821 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2881 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2941 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3001 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3061 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3121 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3181 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3241 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3301 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3361 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3601 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4141 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4201 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4261 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     4321 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4381 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4441 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4501 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4561 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4621 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4681 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4741 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4801 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4861 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4921 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4981 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5041 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5101 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5161 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     5221 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     5281 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     5341 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5401 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5461 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5521 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5581 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5641 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5701 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     5761 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     5821 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     5881 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5941 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6001 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     6061 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     6121 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     6181 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     6241 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     6301 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6361 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6421 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6481 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6541 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcac
//
