LOCUS       sequence-438864-        6940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of GMIP pLxIS domain in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6940)
  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 6940)
  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-GMIP(pLxIS)
FEATURES             Location/Qualifiers
     source          1..6940
                     /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             1634..1651
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1704..1722)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1718..1765
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1802..2257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1889..1908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2099..2120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2349..3164)
                     /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(2349..3164)
                     /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     3072..3091
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3156..3175
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3286..3874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3775..3794
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4028..4045
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4060..4199
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4125..4144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4285..4307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4301..4492)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5301..6383)
                     /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     6345..6364
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6384..6461)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6667..6686
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6770..6789
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6770..6788
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6789..6813
                     /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             6828..6850
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6869..6886
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6896..6913
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6920..6940
                     /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 gggctatgga ctcagcggag acagagctaa ccccagctcc tgagggcagg
     1381 aagagataca gtgacatctt ccagagcctg gacaacctcg agatatcatt ggggaacgta
     1441 acttttgatc ccctggctgg agaccctgta cgcagagaag acctggagcc tgacaaagct
     1501 gacacagcca cagtggtgac tgaggagaac agtgaagcca gtagttggcg ggatctctcc
     1561 ccagaaggtc ctgcacccct cacagagtga taagaattcg agctccgtcg acaagcttgc
     1621 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1681 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1741 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1801 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1861 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1921 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1981 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2041 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2101 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2161 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2221 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2281 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2341 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2401 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2461 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2521 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2581 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2641 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2701 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2761 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2821 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2881 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2941 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3001 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3061 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3121 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3181 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     3241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3421 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3661 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3721 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3781 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3841 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3901 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3961 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4021 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4081 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4141 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4201 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4261 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4321 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4381 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4441 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4501 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4561 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4621 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4681 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4741 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4801 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4861 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4921 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4981 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5041 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5101 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5161 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5221 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5281 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5401 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5461 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5521 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5581 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5641 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5701 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5761 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5821 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5881 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5941 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6001 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6061 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6121 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6181 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6241 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6301 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6361 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6421 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6481 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6541 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6601 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6661 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6721 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6781 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     6841 aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt
     6901 gccgcgcggc agccatatgg aaaacctgta ttttcagggc
//
