LOCUS       sequence-438975-        7075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of IRSp53 pLxIS domain in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7075)
  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 7075)
  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-IRSp53(pLxIS)
FEATURES             Location/Qualifiers
     source          1..7075
                     /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             1769..1786
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1839..1857)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1853..1900
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1937..2392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2024..2043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2234..2255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2484..3299)
                     /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(2484..3299)
                     /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     3207..3226
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3291..3310
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3421..4009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3910..3929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4163..4180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4195..4334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4260..4279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4420..4442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4436..4627)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5436..6518)
                     /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     6480..6499
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6519..6596)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6802..6821
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6905..6924
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6905..6923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6924..6948
                     /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             6963..6985
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7004..7021
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7031..7048
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7055..7075
                     /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 acgcgcggtg 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 gggctatggc caatagcaat ggctccatcc ttccaagtgc cctgtcagct
     1381 tccaagtcca acctggtcat ctcagatccc attcctggag ccaagccctt gccagtgcca
     1441 cctgagctgg caccatttgt ggggcggatg tctgctcagg agaatgtacc tgtcatgaat
     1501 ggcgtcgcag gcccggacag cgaggactac aacccctggg ctgaccgaaa ggctgcccag
     1561 cccaaatccc tgtctccccc gcagtctcaa agcaagctga gcgactcgta ctccaacaca
     1621 ctccccgtgc gcaagagcgt gacgccgaag aacagctatg ccaccactga gaacaagaca
     1681 ctgccacgtt caagctccat ggcagctggc ctagaacgta attgataaga attcgagctc
     1741 cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
     1801 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1861 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1921 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     2401 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2461 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     2521 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     2581 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     2641 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     2701 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     2761 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2821 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2881 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2941 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     3001 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     3061 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     3121 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     3181 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     3241 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     3301 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     3361 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3421 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3481 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3541 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3601 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3661 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3721 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3781 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3841 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3901 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3961 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4021 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4081 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4141 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4201 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4261 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4321 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4381 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4441 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4501 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4561 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4621 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4681 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4741 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4801 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4861 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4921 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4981 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5041 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     5101 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5161 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5221 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5281 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5341 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5401 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5461 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5521 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5581 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5641 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5701 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5761 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5821 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5881 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5941 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     6001 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     6061 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     6121 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     6181 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     6241 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     6301 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6361 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6421 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6481 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6541 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6601 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6661 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6721 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6781 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6841 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6901 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     6961 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     7021 cagcagcggc ctggtgccgc gcggcagcca tatggaaaac ctgtattttc agggc
//
