LOCUS       sequence-438969-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of TRIF Nterm domain in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  AUTHORS   Landau LM, Chaudhary N, Tien YC, Rogozinska M, Joshi S, Yao C, 
            Crowley J, Hullahalli K, Campbell IW, Waldor MK, Haigis M, Kagan JC
  TITLE     pLxIS-containing domains are biochemically flexible regulators of 
            interferons and metabolism.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2436-2454.e10. doi: 
            10.1016/j.molcel.2024.05.030. Epub 2024 Jun 25.
  PUBMED    38925114
REFERENCE   2  (bases 1 to 7354)
  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"; doi: 
            "10.1016/j.molcel.2024.05.030"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2436-2454.e10"
COMMENT     Sequence Label: pET28a-FKBP-TRIF(Nterm)
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2048..2065
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2118..2136)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2132..2179
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2216..2671
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2303..2322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2513..2534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2763..3578)
                     /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(2763..3578)
                     /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     3486..3505
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3570..3589
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3700..4288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4189..4208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4442..4459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4474..4613
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4539..4558)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4699..4721
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4715..4906)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5715..6797)
                     /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     6759..6778
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6798..6875)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7081..7100
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7184..7203
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7184..7202
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7203..7227
                     /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             7242..7264
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7283..7300
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7310..7327
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7334..7354
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 ggatccgcca ccatgggcgt tcaagtagaa acaatatctc caggagacgg gcgtacattt
       61 cctaagcgtg gccaaacttg cgtagtccat tataccggca tgctggagga cggtaagaag
      121 gtcgatagct ctagagaccg taacaaacct ttcaaattta tgctggggaa acaggaggtt
      181 atccgtggtt gggaggaagg cgtggcccaa atgtccgttg ggcagcgtgc taagttaacc
      241 atctcgccag attacgcata tggggctaca ggccatcctg ggatcattcc cccgcatgcg
      301 acacttgtgt ttgatgtaga gctgttgaag ttggagacgc ggggagttca agtcgaaacg
      361 atttcaccag gtgatggacg cacctttcct aaacggggac aaacgtgcgt cgtgcattac
      421 accggtatgc tggaagacgg caagaaagtc gattcaagcc gcgatagaaa taaacccttc
      481 aaattcatgc ttggaaaaca ggaagtaata cgcggatggg aagagggtgt ggcgcaaatg
      541 tctgtcgggc aaagagctaa actgacaata agtcctgatt acgcgtacgg tgcgactggg
      601 caccctggta tcattccgcc gcacgcgaca cttgtcttcg acgtcgagtt acttaagctt
      661 gagacgcggg gggtgcaggt agagactatt tcgcccggtg acggaagaac tttcccaaag
      721 cggggacaga cctgtgttgt ccactacact gggatgttgg aggatggtaa gaaagttgac
      781 tcaagtcgtg atcgcaacaa accctttaag tttatgcttg ggaagcagga agtcatcaga
      841 ggttgggaag aaggcgtagc tcagatgtca gtgggacaac gggccaaatt aaccatcagt
      901 ccagactacg cctacggagc caccggtcac ccagggatca taccacctca cgcaacctta
      961 gtgttcgatg tggaactttt aaagttagag acgcacggtg tacaagtgga aacaatcagt
     1021 ccaggggatg ggagaacatt ccccaaaaga ggccagacgt gtgtcgtgca ctatacgggg
     1081 atgttagaag atgggaaaaa ggtcgattcg tcacgcgacc gcaacaaacc gtttaaattc
     1141 atgttgggaa agcaggaggt gattcgcggc tgggaggaag gtgtggcaca gatgagcgtg
     1201 ggccagagag caaaactgac tatctctcct gattatgcct acggcgcgac gggccaccct
     1261 ggcataattc caccccacgc taccctggta ttcgatgtcg agcttctgaa gcttgagggt
     1321 gctggggctg gggctatgga caatcctgga ccttctttgc ggggggcatt tggcattttg
     1381 ggagcactcg aacgcgatag actgacacat ttgaagcata agttgggcag tctctgcagt
     1441 ggctcacagg aaagcaagtt gctgcacgca atggttctct tggcattggg gcaagatact
     1501 gaggctaggg tcagtcttga atcccttaaa atgaacactg tagctcagtt ggttgcacac
     1561 cagtgggcag atatggagac taccgaaggt cccgaagaac ctcccgatct ctcatggacc
     1621 gttgccaggc tttatcattt gctcgcagag gagaacctct gtcccgccag tactcgggat
     1681 atggcttatc aggtggccct ccgcgatttt gcaagtcaag gagatcatca acttgggcaa
     1741 ctccaaaacg aggcttggga tagatgtagc tcagatatca agggagaccc ctccgggttt
     1801 cagccacttc actctcacca gggttcactt caaccaccta gtgcctcccc tgcagtcact
     1861 cggagccagc ccagacccat tgacaccccc gactggtcct ggggacatac tctccattcc
     1921 accaattcca ctgctagcct cgcttcccac cttgaaatat cacagtcccc cacacttgcc
     1981 tttctgagtt ctcatcacgg atgataagaa ttcgagctcc gtcgacaagc ttgcggccgc
     2041 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     2101 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2161 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     2221 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2281 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2341 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2401 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2461 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2521 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2581 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2641 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2701 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2761 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2821 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2881 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2941 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3001 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3061 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3121 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3181 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3241 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     3301 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3361 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3421 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3481 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3541 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3601 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3661 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3721 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3781 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3841 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3901 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3961 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4021 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4081 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4141 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4201 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4261 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4321 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4381 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4441 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4501 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4561 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4621 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4681 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4741 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4801 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4861 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4921 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4981 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5041 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     5101 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     5161 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     5221 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     5281 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     5341 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     5401 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5461 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5521 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5581 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5641 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5701 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5761 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5821 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5881 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5941 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     6001 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6061 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6121 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6181 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6241 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6301 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6361 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6421 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6481 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6541 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6601 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6661 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6721 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6781 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6841 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6901 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6961 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7021 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7081 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     7141 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     7201 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     7261 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
     7321 cggcagccat atggaaaacc tgtattttca gggc
//
