LOCUS       sequence-457239-        5357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of MSI2 RRM1 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5357)
  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 5357)
  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_RRM1_pGEX
FEATURES             Location/Qualifiers
     source          1..5357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(206..319)
                     /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(329..352)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(359..1012)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(379..401)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    1035..1051
                     /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(1059..1065,1066..1081,1082..1087))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(1189..1716)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     1665..1687
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1679..1696
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1680..1696
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1712..1728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1712..1728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1736..1752
                     /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(1760..1766,1767..1784,1785..1790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(1839..2921)
                     /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     2883..2902
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2922..2999)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(3072..3089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3243..3831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3323..3342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4002..4793,4794..4862))
                     /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     4625..4644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4863..4967)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5035..5053
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     5198..5220
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ctaactcatg gtggggctga cctaatactt tatctacact tgctgggtct gcgaacgtga
       61 cgaaaccgaa gcctctggag cgtttcgtag tgggatctct catgaccata cattctctaa
      121 tttctccaaa tttgctaaaa tagtctctaa ggctatctgg tgaggtctgc cagctcagtc
      181 caccgataaa cattttaccg gatcccggtt cacgctgacc ctgcgggtgg tgttccagac
      241 gagcacgcag ctgttccagt tcaccagcca gaccttcaac aacgtgacca ccacgccaac
      301 cggtggtttt ttcgtccatt aatcccaggg gcccctggaa cagaacttcc agatccgatt
      361 ttggaggatg gtcgccacca ccaaacgtgg cttgccagcc ctgcaaaggc catgctatat
      421 acttgctgga tttcaagtac ttatcaattt gtgggatagc ttcaatacgt tttttaaaac
      481 aaactaattt tgggaacgca tccaggcaca ttgggtccat gtataaaaca acatcaagag
      541 cgtcatacaa catgaagtca ggatgggtta catgatcacc atttaaatat gttttatgac
      601 ataaacgatc ttcgaacatt ttcagcattt caggtagctt gctaagaaaa tcaactttga
      661 gagtttcaaa gtctttacta tatgcaattc tcgaaacacc gtatctaata tccaaaaccg
      721 ctccttcaag cattgaaatc tctgcacgct cttttggaca accacccaac atgttgtgct
      781 tgtcagctat ataacgtatg atggccatag actgtgttaa tttaacatca ccatcaatat
      841 aataaggaag attgggaaac tccaaaccca attcaaactt tttgtttcgc catttatcac
      901 cttcatcgcg ctcatacaaa tgctcttcat atttttcttc aagatattcc aaaagaagtc
      961 gagtgggttg cacaaggccc ttaattttcc aataacctag tataggggac atgaatactg
     1021 tttcctgtgt gaaattgtta tccgctcaca attccacaca ttatacgagc cgatgattaa
     1081 ttgtcaacag ctcatttcag aatatttgcc agaaccgtta tgatgtcggc gcaaaaaaca
     1141 ttatccagaa cgggagtgcg ccttgagcga cacgaattat gcagtgattt acgacctgca
     1201 cagccatacc acagcttccg atggctgcct gacgccagaa gcattggtgc accgtgcagt
     1261 cgataagcta attccaacgc catcaaaaat aattcgcgtc tggccttcct gtagccagct
     1321 ttcatcaaca ttaaatgtga gcgagtaaca acccgtcgga ttctccgtgg gaacaaacgg
     1381 cggattgacc gtaatgggat aggtcacgtt ggtgtagatg ggcgcatcgt aaccgtgcat
     1441 ctgccagttt gaggggacga cgacagtatc ggcctcagga agatcgcact ccagccagct
     1501 ttccggcacc gcttctggtg ccggaaacca ggcaaagcgc cattcgccat tcaggctgcg
     1561 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     1621 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     1681 taaaacgacg gccagtgaat ccgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1741 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1801 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1861 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1921 gtattgggcg ctagcgtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     1981 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     2041 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     2101 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     2161 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     2221 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     2281 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     2341 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     2401 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     2461 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     2521 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     2581 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     2641 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     2701 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     2761 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     2821 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     2881 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     2941 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcg
     3001 gaatttatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3061 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3121 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3181 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3241 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3301 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3361 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3421 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3481 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3541 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3601 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3661 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     3721 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3781 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3841 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3901 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3961 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4021 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4081 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4141 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4201 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4261 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     4321 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4381 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4441 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4501 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4561 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     4621 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4681 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4741 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4801 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4861 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4921 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4981 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5041 cgtatcacga ggccctttcg tcttcaagaa ttatacactc cgctatcgct acgtgactgg
     5101 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5161 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5221 ttttcaccgt catcaccgaa acgcgcgagg cagatcgtca gtcagtcacg atgcggccgc
     5281 tcatttcttt gttcttgtga ccatcttggg ttgcgctcga cgaggaaatg caactttggg
     5341 gtcaatcgtc ttggaat
//
