LOCUS       sequence-456259-        5333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of mouse MSI1 RRM2 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5333)
  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 5333)
  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_RRM2_pGEX
FEATURES             Location/Qualifiers
     source          1..5333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(60..82)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(227..245)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        313..417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(418..486,487..1278)
                     /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(636..655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1449..2037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1938..1957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2191..2208
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2281..2358
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2359..3441
                     /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(2378..2397)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3490..3495,3496..3513,3514..3520)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3528..3544
                     /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     3552..3568
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3552..3568
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3564..4091
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(3584..3601)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3584..3600)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3593..3615)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(4193..4198,4199..4214,4215..4221)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4229..4245
                     /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             4268..4921
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     4879..4901
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             4928..4951
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4961..5074
                     /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"
ORIGIN
        1 cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac
       61 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
      121 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
      181 cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc
      241 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
      301 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      361 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      421 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      481 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      541 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      601 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      661 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      721 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      781 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      841 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      901 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      961 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1021 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1081 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1141 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1201 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1261 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1321 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1381 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1441 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1501 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1561 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1621 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1681 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1741 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1801 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1861 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1921 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1981 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2041 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2101 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2161 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2221 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc
     2281 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     2341 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     2401 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     2461 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     2521 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     2581 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     2641 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     2701 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     2761 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     2821 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     2881 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     2941 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     3001 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     3061 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     3121 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     3181 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     3241 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     3301 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccacgctagc gcccaatacg
     3361 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     3421 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     3481 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     3541 acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac
     3601 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     3661 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     3721 gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa
     3781 gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga
     3841 tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc
     3901 cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa
     3961 gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga
     4021 ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt
     4081 gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat
     4141 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat
     4201 taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     4261 agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg
     4321 acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg
     4381 tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta
     4441 tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa
     4501 gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc
     4561 ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct
     4621 caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg
     4681 tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc
     4741 tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg
     4801 ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta
     4861 tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa
     4921 atcggatctg gaagttctgt tccaggggcc cctgggatta atggacgaaa aaaccaccgg
     4981 ttggcgtggt ggtcacgttg ttgaaggtct ggctggtgaa ctggaacagc tgcgtgctcg
     5041 tctggaacac cacccgcagg gtcagcgtga accgggatcc aagaagatct tcgtgggggg
     5101 gctgtctgtg aacaccacgg tggaagatgt gaaacactat ttcgagcagt tcggaaaggt
     5161 ggatgatgcc atgctgatgt tcgacaaaac caccaacagg cacagagggt ttggatttgt
     5221 cacgtttgag agcgaggaca tcgtagagaa agtttgtgag atccacttcc atgaaatcaa
     5281 caacaaaatg gtggaatgca agaaagccca gccaaaggag gtgatgtcct gag
//
