LOCUS       sequence-456258-        5336 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recombinant expression of mouse MSI1 RRM1 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5336)
  TITLE     Dissecting RNA Selectivity Mediated by Tandem RNA-Binding Domains
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5336)
  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            "Unpublished"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: MSI1_RRM1_pGEX
FEATURES             Location/Qualifiers
     source          1..5336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..195
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        268..345
                     /gene="lacI"
                     /label=lacI promoter
     CDS             346..1428
                     /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(365..384)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1477..1482,1483..1500,1501..1507)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1515..1531
                     /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     1539..1555
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1539..1555
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1551..2078
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1571..1588)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1571..1587)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1580..1602)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2180..2185,2186..2201,2202..2208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2216..2232
                     /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             2255..2908
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2866..2888
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2915..2938
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2948..3061
                     /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"
     primer_bind     complement(3383..3405)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3550..3568)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3636..3740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3741..3809,3810..4601)
                     /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(3959..3978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4772..5336,1..24)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5261..5280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
       61 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
      121 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
      181 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
      241 tgcggtattt cacaccgcat aaattccgac accatcgaat ggcgcaaaac ctttcgcggt
      301 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
      361 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
      421 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
      481 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
      541 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
      601 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
      661 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
      721 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
      781 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
      841 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
      901 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
      961 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     1021 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     1081 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     1141 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     1201 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     1261 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     1321 agaaaaacca cgctagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     1381 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     1441 taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     1501 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     1561 ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     1621 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     1681 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt
     1741 ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata
     1801 ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg
     1861 tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt
     1921 actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt
     1981 ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc
     2041 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
     2101 tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca
     2161 aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
     2221 agcggataac aatttcacac aggaaacagt attcatgtcc cctatactag gttattggaa
     2281 aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga
     2341 agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg
     2401 tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat
     2461 ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg
     2521 tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat
     2581 tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat
     2641 gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac
     2701 ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg
     2761 cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat
     2821 tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac
     2881 gtttggtggt ggcgaccatc ctccaaaatc ggatctggaa gttctgttcc aggggcccct
     2941 gggattaatg gacgaaaaaa ccaccggttg gcgtggtggt cacgttgttg aaggtctggc
     3001 tggtgaactg gaacagctgc gtgctcgtct ggaacaccac ccgcagggtc agcgtgaacc
     3061 gggatcctgc aagatgttca tcggaggact cagttggcag accacgcagg aagggctgcg
     3121 cgaatacttc ggccagttcg gggaggtgaa agagtgtctg gtgatgcggg accccctgac
     3181 caaaagatcc aggggtttcg gcttcgtcac tttcatggac caggcggggg tggataaagt
     3241 gctggcgcaa tcgcggcacg agctcgactc caaaacaatt gaccccaagg tggcctttcc
     3301 tcgaagagca cagcctaagt gagcggccgc atcgtgactg actgacgatc tgcctcgcgc
     3361 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     3421 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     3481 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa
     3541 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3601 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3661 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3721 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3781 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3841 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3901 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3961 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     4021 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4081 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4141 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4201 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4261 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     4321 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4381 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4441 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4501 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4561 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4621 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4681 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4741 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4801 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4861 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4921 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4981 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5041 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5101 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5161 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5221 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5281 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcg
//
