LOCUS       sequence-456246-        6068 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recombinant expression of MSI2 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6068)
  TITLE     Dissecting RNA Selectivity Mediated by Tandem RNA-Binding Domains
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6068)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: MSI2_2xRRM_pGEX
FEATURES             Location/Qualifiers
     source          1..6068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    227..243
                     /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(251..257,258..273,274..279))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(381..908)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     857..879
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     871..888
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     872..888
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(904..920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(904..920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    928..944
                     /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(952..958,959..976,977..982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(1031..2113)
                     /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     2075..2094
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2114..2191)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(2264..2281)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2435..3023)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2515..2534)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3194..3985,3986..4054))
                     /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     3817..3836
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4055..4159)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4227..4245
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     4390..4412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5466..5579)
                     /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(5589..5612)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(5619..6068,1..204))
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(5639..5661)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
ORIGIN
        1 agactgtgtt aatttaacat caccatcaat ataataagga agattgggaa actccaaacc
       61 caattcaaac tttttgtttc gccatttatc accttcatcg cgctcataca aatgctcttc
      121 atatttttct tcaagatatt ccaaaagaag tcgagtgggt tgcacaaggc ccttaatttt
      181 ccaataacct agtatagggg acatgaatac tgtttcctgt gtgaaattgt tatccgctca
      241 caattccaca cattatacga gccgatgatt aattgtcaac agctcatttc agaatatttg
      301 ccagaaccgt tatgatgtcg gcgcaaaaaa cattatccag aacgggagtg cgccttgagc
      361 gacacgaatt atgcagtgat ttacgacctg cacagccata ccacagcttc cgatggctgc
      421 ctgacgccag aagcattggt gcaccgtgca gtcgataagc taattccaac gccatcaaaa
      481 ataattcgcg tctggccttc ctgtagccag ctttcatcaa cattaaatgt gagcgagtaa
      541 caacccgtcg gattctccgt gggaacaaac ggcggattga ccgtaatggg ataggtcacg
      601 ttggtgtaga tgggcgcatc gtaaccgtgc atctgccagt ttgaggggac gacgacagta
      661 tcggcctcag gaagatcgca ctccagccag ctttccggca ccgcttctgg tgccggaaac
      721 caggcaaagc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      781 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
      841 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga atccgtaatc
      901 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
      961 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac ttacattaat
     1021 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1081 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctagcgtg gtttttcttt
     1141 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     1201 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1261 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1321 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1381 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1441 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1501 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1561 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     1621 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     1681 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     1741 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     1801 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     1861 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     1921 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     1981 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     2041 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     2101 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     2161 cgcgaaaggt tttgcgccat tcgatggtgt cggaatttat gcggtgtgaa ataccgcaca
     2221 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     2281 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2341 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2401 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2461 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2521 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2581 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2641 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2701 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2761 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2821 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     2881 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2941 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3001 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3061 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3121 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3181 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3241 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3301 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3361 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3421 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3481 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     3541 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3601 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3661 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3721 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3781 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     3841 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3901 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3961 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4021 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4081 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4141 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4201 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     4261 aattatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4321 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4381 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4441 ggcagatcgt cagtcagtca cgatgcggcc gctcaatggt atccatttgt aaaggccgtt
     4501 gcaatcaaag gtccagctat gccgtggccg aagcccgagc ccggctgtgg gctggccgca
     4561 cttatgtaat tccccactcc ggagtcctgg ctggcagggc cgtagagatc ggcgacaggt
     4621 cctgggctgt tggcccccgg gaagcctccg ggccgcgccg ggttggagcc tgatcctctt
     4681 gctgccgcca ccgccgctgc tgccactggt ccataagccg ctgctgggaa gcctgggaac
     4741 tgatagccat agcttggagc aaatccgggg tagccacggc cataggtcgc cacgaagttg
     4801 ggatatccca gcatccccat gccaagcatg aacgcgtcca tggtgtaagg cagtccccgg
     4861 gcccggcctc ttgtcccagg tgggaacatg acttctttcg gctgagcttt cttacattct
     4921 accattttat tattgatttc atggaaatga atctcacaga ctttctccac aacatcttca
     4981 ttctcaaaag tgacaaagcc aaaccctctg tgcctgttgg tagttttatc aaacatcagc
     5041 attgcatctt ccaccttgcc aaactgctcg aaatattgct ttacatcttc cactactgtg
     5101 ttcgcagata acccgcctac aaatattttc tttgttcttg tgaccatctt gggttgcgct
     5161 cgacgaggaa atgcaacttt ggggtcaatc gtcttggaat ctaactcatg gtggggctga
     5221 cctaatactt tatctacact tgctgggtct gcgaacgtga cgaaaccgaa gcctctggag
     5281 cgtttcgtag tgggatctct catgaccata cattctctaa tttctccaaa tttgctaaaa
     5341 tagtctctaa ggctatctgg tgaggtctgc cagctcagtc caccgataaa cattttaccg
     5401 gggtcgtgct gggagtcgtt ggcgctgccc gaggtgcctt ggctcccatt tgcctccatg
     5461 gatcccggtt cacgctgacc ctgcgggtgg tgttccagac gagcacgcag ctgttccagt
     5521 tcaccagcca gaccttcaac aacgtgacca ccacgccaac cggtggtttt ttcgtccatt
     5581 aatcccaggg gcccctggaa cagaacttcc agatccgatt ttggaggatg gtcgccacca
     5641 ccaaacgtgg cttgccagcc ctgcaaaggc catgctatat acttgctgga tttcaagtac
     5701 ttatcaattt gtgggatagc ttcaatacgt tttttaaaac aaactaattt tgggaacgca
     5761 tccaggcaca ttgggtccat gtataaaaca acatcaagag cgtcatacaa catgaagtca
     5821 ggatgggtta catgatcacc atttaaatat gttttatgac ataaacgatc ttcgaacatt
     5881 ttcagcattt caggtagctt gctaagaaaa tcaactttga gagtttcaaa gtctttacta
     5941 tatgcaattc tcgaaacacc gtatctaata tccaaaaccg ctccttcaag cattgaaatc
     6001 tctgcacgct cttttggaca accacccaac atgttgtgct tgtcagctat ataacgtatg
     6061 atggccat
//
