LOCUS       sequence-335640-        7583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses human TFIIE. The resulting plasmid was used to generate
            a single expression construct encoding 2 subunits, with His-tag on 
            TF2E1.Originally from MN Gonzalez, was submitted with permission.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7583)
  AUTHORS   He Y, Sato S, Tomomori-Sato C, Chen S, Goode ZH, Conaway JW, Conaway
            RC
  TITLE     Elongin functions as a loading factor for Mediator at 
            ATF6alpha-regulated ER stress response genes.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Sep 28;118(39):e2108751118. doi: 
            10.1073/pnas.2108751118.
  PUBMED    34544872
REFERENCE   2  (bases 1 to 7583)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-09-28"; volume: "118(39):e2108751118. 
            doi"; pages: " 10.1073/pnas.2108751118"
COMMENT     Sequence Label: pETDuet-TFIIE
FEATURES             Location/Qualifiers
     source          1..7583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1528..1546
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1547..1571
                     /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)."
     CDS             2532..2576
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2614..2632)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2628..2675
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2712..3167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3009..3030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3282..4073,4074..4142))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3905..3924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4143..4235)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4316..4904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4805..4824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5058..5075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5090..5229
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5155..5174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5315..5337
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5331..5522)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6094..7176)
                     /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     7138..7157
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7177..7254)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7460..7479
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7567..7583,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acatggcaga cccagatgtc ctcactgaag ttccagcagc
      181 attgaagcgg ttagccaagt atgtgatccg gggattttat ggcattgagc atgccttggc
      241 cttggacatc ttgatcagga actcctgtgt gaaagaggag gatatgctgg agctgctcaa
      301 gtttgatcgg aagcaacttc gatcagtttt gaataattta aagggagaca agtttatcaa
      361 atgcagaatg agggtagaga ctgctgcaga cgggaaaacc actcgccata actactactt
      421 catcaattat cgtactcttg ttaatgtggt aaaatataaa ctggaccaca tgagaagaag
      481 aattgagacc gatgagagag attcgaccaa ccgggcttcc ttcaaatgtc ctgtctgtag
      541 tagtactttc acagacttag aagctaatca gctctttgat cctatgacag gaactttccg
      601 ctgtactttt tgccatacag aggtagaaga ggatgaatca gcaatgccca aaaaagatgc
      661 acgcacactt ttggcaaggt ttaatgaaca aattgagccc atttatgcat tgcttcggga
      721 gacagaggat gtgaacttgg cctatgaaat acttgagcca gaacccacag aaatcccagc
      781 cctgaaacag agcaaggacc atgcagcaac tactgctgga gctgctagcc tagcaggtgg
      841 gcaccaccgg gaagcatggg ccaccaaagg tccttcctat gaagacttat acactcagaa
      901 tgttgtcatt aacatggatg accaagaaga tcttcatcga gcctcactgg aagggaaatc
      961 tgccaaagag aggcctattt ggttgagaga aagcactgtc caaggggcat atggttctga
     1021 agatatgaaa gaagggggca tagatatgga cgcatttcag gagcgtgagg aaggccatgc
     1081 tgggcctgat gacaacgaag aggtcatgcg agcactgctc attcacgaga aaaagacttc
     1141 ctctgccatg gctggttcag tgggggcagc tgctccagtg accgctgcca atggcagtga
     1201 ctcagaaagc gagaccagtg agtcagatga tgattctcca ccccgtccgg cagctgtggc
     1261 tgtgcataaa cgagaagagg atgaagagga agatgacgag tttgaagaag tagcagatga
     1321 ccccattgtc atggtggctg gccgtccgtt ctcctacagt gaagtgagcc aacggccaga
     1381 gctagtggcc cagatgacac cagaagaaaa ggaagcatat atagcaatgg gacaacgcat
     1441 gtttgaggac ctctttgagt gagcggccgc ataatgctta agtcgaacag aaagtaatcg
     1501 tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1561 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata catatggcag
     1621 atctcaattg gatatcaatg gatccaagcc tgttgagaga aagggagctg ttcaaaaaac
     1681 gagctctttc tactcctgta gtagaaaaac gttcagcatc ttctgagtca tcatcatcat
     1741 cgtcaaagaa gaagaaaaca aaggtagaac atggaggatc gtcaggctct aaacaaaatt
     1801 ctgatcatag caatggatca tttaacttga aagctttgtc aggaagctct ggatataagt
     1861 ttggtgttct tgctaagatt gtgaattaca tgaagacacg gcatcagcga ggagatacgc
     1921 atcctctaac cttagatgaa attttggatg aaacacaaca tttagatatt ggactcaagc
     1981 agaaacaatg gctaatgact gaggctttag tcaacaatcc caaaattgaa gtaatagatg
     2041 ggaagtatgc tttcaagccc aagtacaacg tgagagataa gaaggcccta cttaggctct
     2101 tagatcagca tgaccagcga ggattaggag gaattctttt agaagacata gaagaagcac
     2161 tgcccaattc ccagaaagct gtcaaggctt tgggggacca gatactattt gtaaatcgtc
     2221 ccgataagaa gaaaatactt ttcttcaatg ataagagctg tcagttttct gtggatgaag
     2281 aatttcagaa actgtggagg agtgtcactg tagattccat ggacgaggag aaaattgaag
     2341 aatatctgaa gcgacagggt atttcttcca tgcaggaatc tggaccaaag aaagtggccc
     2401 ctattcagag aaggaaaaag cctgcttcac agaaaaagcg acgctttaag actcataacg
     2461 aacacttggc tggagtgctg aaggattact ctgacattac ttccagcaaa tagggtaccc
     2521 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     2581 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     2641 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2701 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2761 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3061 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3121 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
     3181 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3241 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3301 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3361 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3421 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3481 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3541 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3601 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3661 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3721 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3781 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3841 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3901 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3961 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4021 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4081 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4141 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     4201 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     4261 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4321 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4381 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4441 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4501 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4561 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4621 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4681 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4741 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4801 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4861 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4921 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4981 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5041 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5101 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5161 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5221 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5281 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5341 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5401 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5461 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5521 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5581 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5641 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5701 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5761 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     5821 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     5881 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     5941 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     6001 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     6061 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6121 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6181 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     6241 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     6301 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6361 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6421 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6481 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6541 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6601 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6661 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6721 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6781 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6841 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6901 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6961 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     7021 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     7081 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     7141 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     7201 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     7261 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     7321 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     7381 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     7441 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     7501 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     7561 cgaaattaat acgactcact ata
//
