LOCUS       sequence-456251-        5318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of MSI2 RRM2 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5318)
  AUTHORS   Harris SE, Hu Y, Bridges K, Cavazos FF Jr, Martyr JG, Guzman BB, 
            Murn J, Aleman MM, Dominguez D
  TITLE     Dissecting RNA selectivity mediated by tandem RNA-binding domains.
  JOURNAL   J Biol Chem. 2025 May;301(5):108435. doi: 10.1016/j.jbc.2025.108435.
            Epub 2025 Mar 20.
  PUBMED    40120682
REFERENCE   2  (bases 1 to 5318)
  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: "J Biol 
            Chem."; date: "2025-05"; volume: "301(5):108435. doi"; pages: " 
            10.1016/j.jbc.2025.108435. Epub 2025 Mar 20"
COMMENT     Sequence Label: MSI2_RRM2_pGEX
FEATURES             Location/Qualifiers
     source          1..5318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    26..42
                     /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)."
     promoter        complement(join(50..56,57..72,73..78))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(180..707)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     656..678
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     670..687
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     671..687
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(703..719)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(703..719)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    727..743
                     /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)."
     promoter        complement(join(751..757,758..775,776..781))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(830..1912)
                     /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     1874..1893
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1913..1990)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(2063..2080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2234..2822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2314..2333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2993..3784,3785..3853))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3616..3635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3854..3958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4026..4044
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     4189..4211
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4515..4628)
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             complement(4638..4661)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(4668..5318,1..3))
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(4688..4710)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
ORIGIN
        1 catgaatact gtttcctgtg tgaaattgtt atccgctcac aattccacac attatacgag
       61 ccgatgatta attgtcaaca gctcatttca gaatatttgc cagaaccgtt atgatgtcgg
      121 cgcaaaaaac attatccaga acgggagtgc gccttgagcg acacgaatta tgcagtgatt
      181 tacgacctgc acagccatac cacagcttcc gatggctgcc tgacgccaga agcattggtg
      241 caccgtgcag tcgataagct aattccaacg ccatcaaaaa taattcgcgt ctggccttcc
      301 tgtagccagc tttcatcaac attaaatgtg agcgagtaac aacccgtcgg attctccgtg
      361 ggaacaaacg gcggattgac cgtaatggga taggtcacgt tggtgtagat gggcgcatcg
      421 taaccgtgca tctgccagtt tgaggggacg acgacagtat cggcctcagg aagatcgcac
      481 tccagccagc tttccggcac cgcttctggt gccggaaacc aggcaaagcg ccattcgcca
      541 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      601 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      661 tcacgacgtt gtaaaacgac ggccagtgaa tccgtaatca tggtcatagc tgtttcctgt
      721 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      781 agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      841 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      901 aggcggtttg cgtattgggc gctagcgtgg tttttctttt caccagtgag acgggcaaca
      961 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1021 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1081 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1141 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1201 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1261 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1321 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1381 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1441 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1501 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1561 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1621 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1681 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1741 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1801 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1861 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1921 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     1981 cgatggtgtc ggaatttatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     2041 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2101 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2161 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2221 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2281 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2341 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2401 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2461 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2521 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2581 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2641 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     2701 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2761 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2821 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2881 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2941 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3001 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3061 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3121 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3181 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3241 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3301 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3361 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3421 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3481 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3541 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3601 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3661 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3721 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3781 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3841 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3901 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3961 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4021 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attatacact ccgctatcgc
     4081 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4141 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4201 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagatcgtc agtcagtcac
     4261 gatgcggccg ctcacggctg agctttctta cattctacca ttttattatt gatttcatgg
     4321 aaatgaatct cacagacttt ctccacaaca tcttcattct caaaagtgac aaagccaaac
     4381 cctctgtgcc tgttggtagt tttatcaaac atcagcattg catcttccac cttgccaaac
     4441 tgctcgaaat attgctttac atcttccact actgtgttcg cagataaccc gcctacaaat
     4501 attttcttgg atcccggttc acgctgaccc tgcgggtggt gttccagacg agcacgcagc
     4561 tgttccagtt caccagccag accttcaaca acgtgaccac cacgccaacc ggtggttttt
     4621 tcgtccatta atcccagggg cccctggaac agaacttcca gatccgattt tggaggatgg
     4681 tcgccaccac caaacgtggc ttgccagccc tgcaaaggcc atgctatata cttgctggat
     4741 ttcaagtact tatcaatttg tgggatagct tcaatacgtt ttttaaaaca aactaatttt
     4801 gggaacgcat ccaggcacat tgggtccatg tataaaacaa catcaagagc gtcatacaac
     4861 atgaagtcag gatgggttac atgatcacca tttaaatatg ttttatgaca taaacgatct
     4921 tcgaacattt tcagcatttc aggtagcttg ctaagaaaat caactttgag agtttcaaag
     4981 tctttactat atgcaattct cgaaacaccg tatctaatat ccaaaaccgc tccttcaagc
     5041 attgaaatct ctgcacgctc ttttggacaa ccacccaaca tgttgtgctt gtcagctata
     5101 taacgtatga tggccataga ctgtgttaat ttaacatcac catcaatata ataaggaaga
     5161 ttgggaaact ccaaacccaa ttcaaacttt ttgtttcgcc atttatcacc ttcatcgcgc
     5221 tcatacaaat gctcttcata tttttcttca agatattcca aaagaagtcg agtgggttgc
     5281 acaaggccct taattttcca ataacctagt atagggga
//
