LOCUS       sequence-351371-        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-OsTPL(1-209)
FEATURES             Location/Qualifiers
     source          1..6325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     929..948
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(968..1045)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1251..1270
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1354..1373
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1354..1372
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1373..1397
                     /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             1412..1434
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1447..1464
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1468..1758
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             2410..2427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2480..2498)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2494..2541
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2578..3033
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2665..2684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2875..2896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3059..3163
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3164..3232,3233..4024)
                     /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(3382..3401)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4195..4783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4684..4703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4937..4954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4969..5108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5034..5053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5194..5216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5210..5401)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6210..6325,1..967))
                     /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 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
       61 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
      121 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
      181 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
      241 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
      301 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
      361 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
      421 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
      481 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
      541 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
      601 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
      661 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
      721 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
      781 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
      841 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
      901 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
      961 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     1021 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     1081 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     1141 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     1201 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     1261 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     1321 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     1381 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
     1441 atgggtcatc accatcatca tcacgggtcg gactcagaag tcaatcaaga agctaagcca
     1501 gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc cgatggatct
     1561 tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat ggaagcgttc
     1621 gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg tattagaatt
     1681 caagctgatc aggcccctga agatttggac atggaggata acgatattat tgaggctcac
     1741 cgcgaacaga ttggaggtgg atccatgagt agcctgagtc gcgagctggt gttcctgatc
     1801 ttacaattcc tggacgagga gaagttcaag gagacagtgc acaagctgga gcaggagagc
     1861 ggcttctact tcaacatgaa gtacttcgag gacgaggtga tcaacggcaa ctgggacgag
     1921 gtggaacgtt acctgggcgg cttcacaaag gtggacgaca accgctacag catgaaaatc
     1981 ttctttgaga tccgcaagca gaagtacctg gaggccctgg acaagcacga tcgcagtaag
     2041 gccgtggaga ttctggtgaa ggacctgaag gtgttcgcaa gtttcaacga ggagctgttc
     2101 aaggagatca cccagctgct gaccctggag aacttccgcg agaatgagca gctgagcaag
     2161 tatggcgaca ccaaaagcgc ccgcgccatc atgctggtgg agctgaagaa actgattgag
     2221 gccaaccctc tgttccgcga taagttacaa ttcccgaacc tgaagagcag ccgcctgcgc
     2281 accctgatca accagagcct gaactggcag catcagctgt gcaaaaaccc gcgcccgaat
     2341 ccggacatca agaccctgtt cgtggatcac agctgtggcc agccgaatgg ctaagcggcc
     2401 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     2461 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     2521 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     2581 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2641 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2701 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2761 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2821 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2881 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2941 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3001 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     3061 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3121 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3181 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3241 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3301 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3361 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3421 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3481 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3541 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3601 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3661 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     3721 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3781 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3841 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta tcattgcagc
     3901 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3961 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4021 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4081 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4141 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4201 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4261 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4321 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4381 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4441 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4501 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4561 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4621 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4681 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4741 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4801 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4861 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4921 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4981 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     5041 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     5101 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5161 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5221 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     5281 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     5341 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     5401 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     5461 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     5521 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     5581 gccagcgctt cgtttataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     5641 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     5701 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     5761 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     5821 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     5881 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5941 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     6001 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     6061 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     6121 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     6181 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6241 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6301 gccagggtgg tttttctttt cacca
//
