LOCUS       sequence-457948-        6170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of mouse MSI1 Swap with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6170)
  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 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"; 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_SWAP_2xRRM_pGEX
FEATURES             Location/Qualifiers
     source          1..6170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(59..64,65..82,83..89)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    97..113
                     /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     121..137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     121..137
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             133..660
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(153..170)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(153..169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(162..184)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(762..767,768..783,784..790)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    798..814
                     /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             837..1490
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1448..1470
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1497..1520
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1530..1643
                     /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      2464..2473
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2799..2821)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2966..2984)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3052..3156
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3157..3225,3226..4017)
                     /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(3375..3394)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4188..4776
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4677..4696
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4930..4947
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5020..5097
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5098..6170,1..10)
                     /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(5117..5136)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccccaggctt
       61 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      121 caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac
      181 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
      241 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
      301 ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag
      361 tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac
      421 gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc
      481 acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag
      541 gaaggccaga cgcgaattat ttttgatggc gttggaatta gcttatcgac tgcacggtgc
      601 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
      661 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
      721 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc
      781 tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca gtattcatgt
      841 cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg
      901 aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc
      961 gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg
     1021 atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt
     1081 tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata
     1141 ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt
     1201 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
     1261 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
     1321 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
     1381 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
     1441 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg
     1501 aagttctgtt ccaggggccc ctgggattaa tggacgaaaa aaccaccggt tggcgtggtg
     1561 gtcacgttgt tgaaggtctg gctggtgaac tggaacagct gcgtgctcgt ctggaacacc
     1621 acccgcaggg tcagcgtgaa ccgggatcca tggagactga cgcgccccag cccggcctcg
     1681 cctccccgga ctcgccgcac gaccccaaga agatcttcgt gggggggctg tctgtgaaca
     1741 ccacggtgga agatgtgaaa cactatttcg agcagttcgg aaaggtggat gatgccatgc
     1801 tgatgttcga caaaaccacc aacaggcaca gagggtttgg atttgtcacg tttgagagcg
     1861 aggacatcgt agagaaagtt tgtgagatcc acttccatga aatcaacaac aaaatggtgg
     1921 aatgcaagaa agcccagcca aaggaggtga tgtccatggt cactcggacg tgcaagatgt
     1981 tcatcggagg actcagttgg cagaccacgc aggaagggct gcgcgaatac ttcggccagt
     2041 tcggggaggt gaaagagtgt ctggtgatgc gggaccccct gaccaaaaga tccaggggtt
     2101 tcggcttcgt cactttcatg gaccaggcgg gggtggataa agtgctggcg caatcgcggc
     2161 acgagctcga ctccaaaaca attgacccca aggtggcctt tcctcgaaga gcacagccta
     2221 agccgacagg ctcagcccgg ggcaggtctc gggtcatgcc ctacggaatg gatgccttca
     2281 tgctgggtat tgggatgctg ggttacccag ggttccaagc cacgacctac gccagccgga
     2341 gttacacagg ccttgcccct ggttacacct accagttccc cgaattccgt gtagagcgga
     2401 gccctctccc gagcgcccca gtcctccccg agctcacagc tatccctctc acggcttatg
     2461 ggcccatggc ggcggcagcg gcggcggcag ctgtagttcg agggacaggc tctcacccct
     2521 ggacgatggc tccccctcca ggttccactc ccagccgcac agggggcttc ctagggacca
     2581 caagccccgg ccccatggct gagctctacg gggcagccaa ccaggactcc ggggtcagca
     2641 gttacatcag cgccgccagc cccgccccca gcactggttt cggccacagt cttgggggtc
     2701 ccttgattgc cacagccttc accaatgggt accactgagc ggccgcatcg tgactgactg
     2761 acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     2821 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     2881 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     2941 gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3001 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3061 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3121 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3181 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3241 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3301 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3361 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3421 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3481 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3541 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3601 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3661 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3721 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3781 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3841 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3901 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3961 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4021 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4081 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4141 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4201 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4261 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4321 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4381 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4441 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4501 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4561 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4621 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4681 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4741 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4801 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4861 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4921 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4981 cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggcgcaaa
     5041 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     5101 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     5161 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     5221 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     5281 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     5341 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     5401 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     5461 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     5521 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     5581 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     5641 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     5701 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     5761 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     5821 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     5881 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     5941 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     6001 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     6061 tcactggtga aaagaaaaac cacgctagcg cccaatacgc aaaccgcctc tccccgcgcg
     6121 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
//
