LOCUS       sequence-192691-        6055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses lambdaN+-L+-GST fusion protein for affinity purification 
            with ARiBo method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6055)
  AUTHORS   Di Tomasso G, Lampron P, Dagenais P, Omichinski JG, Legault P
  TITLE     The ARiBo tag: a reliable tool for affinity purification of RNAs 
            under native conditions.
  JOURNAL   Nucleic Acids Res. 2011 Feb;39(3):e18. doi: 10.1093/nar/gkq1084. 
            Epub 2010 Nov 11.
  PUBMED    21071425
REFERENCE   2  (bases 1 to 6055)
  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: "Nucleic 
            Acids Res."; date: "2011-02"; volume: "39(3):e18. doi"; pages: " 
            10.1093/nar/gkq1084. Epub 2010 Nov 11"
COMMENT     Sequence Label: pET42a-lambdaN+-L+-GSH
FEATURES             Location/Qualifiers
     source          1..6055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     728..747
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(767..844)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1050..1069
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1176..1200
                     /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             1215..1237
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1374..2027
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1985..2007
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2055..2072
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2082..2099
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2115..2159
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2190..2201
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2293..2316
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2379..2397)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2393..2440
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2477..2932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2564..2583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2774..2795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3024..3839)
                     /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(3024..3839)
                     /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     3747..3766
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3831..3850
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3961..4549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4450..4469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4703..4720
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4735..4874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4800..4819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4960..4982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4976..5167)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5739..6055,1..766))
                     /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 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
       61 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      121 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      181 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      241 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
      301 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
      361 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
      421 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
      481 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
      541 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
      601 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
      661 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
      721 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
      781 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
      841 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
      901 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
      961 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     1021 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     1081 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
     1141 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     1201 cctctagaaa taattttgtt taactttaag aaggagatat acatatgggg aatgcaaaaa
     1261 cacgccgccg cgaacgtcgc gcagagaaac aggctcaatg gaaagcagca aatggcgcag
     1321 gtgcaggcgc aggtgcaggc gcaggtgcag gcgcaggtgc aggcgcaggt gcaatgtccc
     1381 ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat
     1441 atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa
     1501 acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg
     1561 ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg
     1621 gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta
     1681 gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc
     1741 ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt
     1801 taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt
     1861 tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta
     1921 ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt
     1981 tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatggttaaa
     2041 ctagtggttc tggtcatcac catcaccatc actccgcggg tctggtgcca cgcggtagta
     2101 ctgcaattgg tatgaaagaa accgctgctg ctaaattcga acgccagcac atggacagcc
     2161 cagatctggg taccggtggt ggctccggta ttgagggacg cgggtccatg ggatatcggg
     2221 gatccgaatt ctgtacaggc cttggcgcgc ctgcaggcga gctccgtcga caagcttgcg
     2281 gccgcactcg agcaccacca ccaccaccac caccactaat tgattaatac ctaggctgct
     2341 aaacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     2401 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     2461 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2521 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2581 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2641 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2701 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2761 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2821 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2881 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     2941 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3001 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     3061 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     3121 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     3181 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     3241 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     3301 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     3361 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     3421 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     3481 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     3541 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     3601 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     3661 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     3721 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     3781 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     3841 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     3901 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3961 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4021 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4081 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4141 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4201 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4261 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4321 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4381 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4441 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4501 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4561 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4621 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4681 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4741 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4801 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4861 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4921 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4981 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5041 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5101 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5161 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5221 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5281 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5341 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5401 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcatt tccagacttt
     5461 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5521 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5581 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     5641 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5701 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5761 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5821 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5881 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5941 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     6001 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatgg
//
