LOCUS       sequence-350030-        6325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of TPL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6325)
  AUTHORS   Ke J, Ma H, Gu X, Thelen A, Brunzelle JS, Li J, Xu HE, Melcher K
  TITLE     Structural basis for recognition of diverse transcriptional 
            repressors by the TOPLESS family of corepressors.
  JOURNAL   Sci Adv. 2015 Jul 24;1(6):e1500107. doi: 10.1126/sciadv.1500107. 
            eCollection 2015 Jul.
  PUBMED    26601214
REFERENCE   2  (bases 1 to 6325)
  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: "Sci Adv."; 
            date: "2015-07-24"; volume: "1(6):e1500107. doi"; pages: " 
            10.1126/sciadv.1500107. eCollection 2015 Jul"
COMMENT     Sequence Label: pSumo-His6- Sumo-AtTPL(1-209)
FEATURES             Location/Qualifiers
     source          1..6325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1483..1500
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1504..1794
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             2446..2463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2516..2534)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2530..2577
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2614..3069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2701..2720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2911..2932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3095..3199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3200..3268,3269..4060)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3418..3437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4231..4819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4720..4739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4973..4990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5005..5144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5070..5089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5230..5252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5246..5437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6246..6325,1..1003))
                     /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"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tataccatgg gtcatcacca tcatcatcac
     1501 gggtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
     1561 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
     1621 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
     1681 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcaggc ccctgaagat
     1741 ttggacatgg aggataacga tattattgag gctcaccgcg aacagattgg aggtggatcc
     1801 atgagcagcc tgagccgcga actggtgttc ctgatcttac aattcctgga cgaggagaag
     1861 ttcaaggaga ccgtgcacaa gctggagcag gagagtggtt tcttctttaa catgaagtac
     1921 ttcgaagacg aagtgcacaa cggcaactgg gacgaggtgg agaaatacct gagcggcttc
     1981 accaaggttg acgacaaccg ctatagcatg aagattttct tcgagatccg caagcagaag
     2041 tacctggagg ccctggacaa gcacgatcgc ccgaaggccg tggacatcct ggtgaaggac
     2101 ctgaaggtgt tcagcacctt caacgaagag ctgttcaagg agatcaccca actgctgacc
     2161 ctggagaact tccgcgagaa tgagcagctg agcaagtacg gcgacacaaa gagcgcccgt
     2221 gccatcatgc tggtggagct gaagaaactg atcgaggcaa acccgctgtt ccgcgacaag
     2281 ttacaattcc cgacactgcg caacagccgc ctgcgtaccc tgatcaacca gagcctgaac
     2341 tggcagcacc agctgtgcaa gaacccgcgc ccgaacccgg atatcaagac cctgttcgtg
     2401 gaccacagtt gcggccctcc gaacggttaa gcggccgcac tcgagcacca ccaccaccac
     2461 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2521 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2581 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2761 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2821 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2881 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2941 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3001 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3061 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3121 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3181 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3241 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3301 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3361 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3421 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3481 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3541 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3601 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3661 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3721 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3781 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3841 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3901 agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3961 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4021 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4081 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4141 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4201 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4261 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4321 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4381 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4441 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4501 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4561 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4621 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4681 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4741 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4801 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4861 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4921 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4981 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5041 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5101 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5161 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5221 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5281 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5341 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5401 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5461 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5521 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5581 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5641 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5701 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5761 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5821 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5881 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5941 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     6001 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     6061 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6121 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6181 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6241 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6301 gccaacgcgc ggggagaggc ggttt
//
