LOCUS       sequence-429692-        6170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of mouse Msi1 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6170)
  AUTHORS   Dominguez D, Freese P, Alexis MS, Su A, Hochman M, Palden T, Bazile 
            C, Lambert NJ, Van Nostrand EL, Pratt GA, Yeo GW, Graveley BR, Burge
            CB
  TITLE     Sequence, Structure, and Context Preferences of Human RNA Binding 
            Proteins.
  JOURNAL   Mol Cell. 2018 Jun 7;70(5):854-867.e9. doi: 
            10.1016/j.molcel.2018.05.001. Epub 2018 Jun 7.
  PUBMED    29883606
REFERENCE   2  (bases 1 to 6170)
  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.2018.05.001"; journalName: "Mol Cell"; date: 
            "2018-06-7- 7"; volume: "70"; issue: "5"; pages: "854-867.e9"
COMMENT     Sequence Label: Msi1_2xRRM_pGEX
FEATURES             Location/Qualifiers
     source          1..6170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     603..625
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             652..675
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             685..798
                     /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"
     regulatory      1619..1628
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1954..1976)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2121..2139)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2207..2311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2312..2380,2381..3172)
                     /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     complement(2530..2549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3343..3931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3832..3851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4085..4102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4175..4252
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4253..5335
                     /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     complement(4272..4291)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5384..5389,5390..5407,5408..5414)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5422..5438
                     /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)."
     primer_bind     5446..5462
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5446..5462
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5458..5985
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5478..5495)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5478..5494)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5487..5509)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6087..6092,6093..6108,6109..6115)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6123..6139
                     /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)."
     CDS             join(6162..6170,1..645)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
ORIGIN
        1 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat
       61 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac
      121 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt
      181 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt
      241 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga
      301 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt
      361 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta
      421 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta
      481 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt
      541 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg
      601 cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctggaagtt
      661 ctgttccagg ggcccctggg attaatggac gaaaaaacca ccggttggcg tggtggtcac
      721 gttgttgaag gtctggctgg tgaactggaa cagctgcgtg ctcgtctgga acaccacccg
      781 cagggtcagc gtgaaccggg atccatggag actgacgcgc cccagcccgg cctcgcctcc
      841 ccggactcgc cgcacgaccc ctgcaagatg ttcatcggag gactcagttg gcagaccacg
      901 caggaagggc tgcgcgaata cttcggccag ttcggggagg tgaaagagtg tctggtgatg
      961 cgggaccccc tgaccaaaag atccaggggt ttcggcttcg tcactttcat ggaccaggcg
     1021 ggggtggata aagtgctggc gcaatcgcgg cacgagctcg actccaaaac aattgacccc
     1081 aaggtggcct ttcctcgaag agcacagcct aagatggtca ctcggacgaa gaagatcttc
     1141 gtgggggggc tgtctgtgaa caccacggtg gaagatgtga aacactattt cgagcagttc
     1201 ggaaaggtgg atgatgccat gctgatgttc gacaaaacca ccaacaggca cagagggttt
     1261 ggatttgtca cgtttgagag cgaggacatc gtagagaaag tttgtgagat ccacttccat
     1321 gaaatcaaca acaaaatggt ggaatgcaag aaagcccagc caaaggaggt gatgtccccg
     1381 acaggctcag cccggggcag gtctcgggtc atgccctacg gaatggatgc cttcatgctg
     1441 ggtattggga tgctgggtta cccagggttc caagccacga cctacgccag ccggagttac
     1501 acaggccttg cccctggtta cacctaccag ttccccgaat tccgtgtaga gcggagccct
     1561 ctcccgagcg ccccagtcct ccccgagctc acagctatcc ctctcacggc ttatgggccc
     1621 atggcggcgg cagcggcggc ggcagctgta gttcgaggga caggctctca cccctggacg
     1681 atggctcccc ctccaggttc cactcccagc cgcacagggg gcttcctagg gaccacaagc
     1741 cccggcccca tggctgagct ctacggggca gccaaccagg actccggggt cagcagttac
     1801 atcagcgccg ccagccccgc ccccagcact ggtttcggcc acagtcttgg gggtcccttg
     1861 attgccacag ccttcaccaa tgggtaccac tgagcggccg catcgtgact gactgacgat
     1921 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     1981 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2041 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     2101 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2161 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2221 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2281 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2341 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2401 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2461 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2521 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     2581 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2641 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2701 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2761 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2821 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2881 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2941 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3001 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3061 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3121 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3181 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3241 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3301 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3361 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3421 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3481 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3541 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3601 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3661 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3721 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3781 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3841 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3901 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3961 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4021 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4081 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4141 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt
     4201 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4261 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4321 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4381 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4441 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4501 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4561 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4621 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4681 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4741 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4801 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4861 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4921 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4981 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     5041 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     5101 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     5161 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     5221 ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5281 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5341 acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     5401 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     5461 accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     5521 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     5581 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt
     5641 gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag
     5701 gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac
     5761 accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg
     5821 ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga
     5881 attatttttg atggcgttgg aattagctta tcgactgcac ggtgcaccaa tgcttctggc
     5941 gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc
     6001 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt
     6061 tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg
     6121 aattgtgagc ggataacaat ttcacacagg aaacagtatt catgtcccct
//
