LOCUS       sequence-456254-        6101 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 6101)
  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 6101)
  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: MSI1_2xRRM_pGEX_Flag_noSBP
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(182..187,188..203,204..210)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    218..234
                     /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             257..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             917..940
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             956..979
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1812..1821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2150..2172)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2317..2335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2403..2507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2508..2576,2577..3368)
                     /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(2726..2745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3539..4127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4028..4047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4281..4298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4371..4448
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4449..5531
                     /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(4468..4487)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5580..5585,5586..5603,5604..5610)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5618..5634
                     /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     5642..5658
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5642..5658
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5654..6101,1..80)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5674..5691)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5674..5690)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5683..5705)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
       61 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      121 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      181 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatctgg aagttctgtt ccaggggccc ctgggattac tcgaggacta
      961 caaagacgat gacgacaagc agcgtgaacc gggatccatg gagactgacg cgccccagcc
     1021 cggcctcgcc tccccggact cgccgcacga cccctgcaag atgttcatcg gaggactcag
     1081 ttggcagacc acgcaggaag ggctgcgcga atacttcggc cagttcgggg aggtgaaaga
     1141 gtgtctggtg atgcgggacc ccctgaccaa aagatccagg ggtttcggct tcgtcacttt
     1201 catggaccag gcgggggtgg ataaagtgct ggcgcaatcg cggcacgagc tcgactccaa
     1261 aacaattgac cccaaggtgg cctttcctcg aagagcacag cctaagatgg tcactcggac
     1321 gaagaagatc ttcgtggggg ggctgtctgt gaacaccacg gtggaagatg tgaaacacta
     1381 tttcgagcag ttcggaaagg tggatgatgc catgctgatg ttcgacaaaa ccaccaacag
     1441 gcacagaggg tttggatttg tcacgtttga gagcgaggac atcgtagaga aagtttgtga
     1501 gatccacttc catgaaatca acaacaaaat ggtggaatgc aagaaagccc agccaaagga
     1561 ggtgatgtcc ccgacaggct cagcccgggg caggtctcgg gtcatgccct acggaatgga
     1621 tgccttcatg ctgggtattg ggatgctggg ttacccaggg ttccaagcca cgacctacgc
     1681 cagccggagt tacacaggcc ttgcccctgg ttacacctac cagttccccg aattccgtgt
     1741 agagcggagc cctctcccga gcgccccagt cctccccgag ctcacagcta tccctctcac
     1801 ggcttatggg cccatggcgg cggcagcggc ggcggcagct gtagttcgag ggacaggctc
     1861 tcacccctgg acgatggctc cccctccagg ttccactccc agccgcacag ggggcttcct
     1921 agggaccaca agccccggcc ccatggctga gctctacggg gcagccaacc aggactccgg
     1981 ggtcagcagt tacatcagcg ccgccagccc cgcccccagc actggtttcg gccacagtct
     2041 tgggggtccc ttgattgcca cagccttcac caatgggtac cactgatagg cggccgcatc
     2101 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     2161 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     2221 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     2281 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     2341 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2401 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2461 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2521 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2581 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2641 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2701 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     2761 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2821 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2881 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2941 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3001 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     3061 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3121 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3181 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3241 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3301 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3361 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3421 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3541 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3601 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3661 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3721 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3781 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3841 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3901 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3961 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4021 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4081 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4141 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4201 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4261 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4321 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg
     4381 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     4441 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     4501 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     4561 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     4621 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     4681 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     4741 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     4801 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     4861 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     4921 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     4981 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     5041 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     5101 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     5161 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     5221 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     5281 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     5341 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     5401 tgttgcccgt ctcactggtg aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct
     5461 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5521 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct
     5581 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5641 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
     5701 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5761 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5821 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
     5881 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
     5941 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
     6001 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     6061 ggaaggccag acgcgaatta tttttgatgg cgttggaatt a
//
