LOCUS       sequence-273991-        5744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of RBM4 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5744)
  AUTHORS   Dominguez D, Freese P, Alexis MS, Su A, Hochman M, Palden T, Bazile 
            C, Lambert NJ, Van Nostrand EL, Pratt GA, Yeo GW, Graveley BR, Burge
            CB
  TITLE     Sequence, Structure, and Context Preferences of Human RNA Binding 
            Proteins.
  JOURNAL   Mol Cell. 2018 Jun 7;70(5):854-867.e9. doi: 
            10.1016/j.molcel.2018.05.001. Epub 2018 Jun 7.
  PUBMED    29883606
REFERENCE   2  (bases 1 to 5744)
  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"; doi: 
            "10.1016/j.molcel.2018.05.001"; journalName: "Mol Cell"; date: 
            "2018-06-7- 7"; volume: "70"; issue: "5"; pages: "854-867.e9"
COMMENT     Sequence Label: RBM4_2xRRM-ZNF_pGEX
FEATURES             Location/Qualifiers
     source          1..5744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..17
                     /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             40..693
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     651..673
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             700..723
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             733..846
                     /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(1576..1598)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1743..1761)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1829..1933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1934..2002,2003..2794)
                     /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(2152..2171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2965..3553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3454..3473
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3707..3724
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3797..3874
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3875..4957
                     /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(3894..3913)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5006..5011,5012..5029,5030..5036)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5044..5060
                     /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     5068..5084
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5068..5084
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5080..5607
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5100..5117)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5100..5116)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5109..5131)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5709..5714,5715..5730,5731..5737)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat actaggttat
       61 tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa
      121 tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa
      181 ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag
      241 tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa
      301 gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg
      361 agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct
      421 gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat
      481 gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca
      541 atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca
      601 caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa
      661 gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggaagttct gttccagggg
      721 cccctgggat taatggacga aaaaaccacc ggttggcgtg gtggtcacgt tgttgaaggt
      781 ctggctggtg aactggaaca gctgcgtgct cgtctggaac accacccgca gggtcagcgt
      841 gaaccgggat ccatggtgaa gctgttcatc ggaaacctgc cccgggaggc tacagagcag
      901 gagattcgct cactcttcga gcagtatggg aaggtgctgg aatgtgacat cattaagaat
      961 tacggctttg tgcacataga agacaagacg gcagctgagg atgccatacg caacctgcac
     1021 cattacaagc ttcatggggt gaacatcaac gtggaagcca gcaagaataa gagcaaaacc
     1081 tcaacaaagt tgcatgtggg caacatcagt cccacctgca ccaataagga gcttcgagcc
     1141 aagtttgagg agtatggtcc ggtcatcgaa tgtgacatcg tgaaagatta tgccttcgta
     1201 cacatggagc gggcagagga tgcagtggag gccatcaggg gccttgataa cacagagttt
     1261 caaggcaaac gaatgcacgt gcagttgtcc accagccggc ttaggactgc gcccgggatg
     1321 ggagaccaga gcggctgcta tcggtgcggg aaagaggggc actggtccaa agagtgtccg
     1381 atagatcgtt caggccgcgt ggcagacttg accgagcaat ataatgagca atacggagca
     1441 gtgcgtacgc cttacaccat gagctatggg gattcattgt attacaacaa cgcgtacgga
     1501 gcgctcgatg cctgagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg
     1561 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     1621 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     1681 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa
     1741 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1801 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1861 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1921 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1981 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2041 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2101 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2161 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     2221 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2281 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2341 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2401 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2461 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2521 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2581 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2641 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2701 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2761 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2821 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2881 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2941 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3001 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3061 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3121 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3181 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3241 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3301 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3361 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3421 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3481 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3541 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3601 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3661 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3721 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3781 acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     3841 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     3901 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     3961 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     4021 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     4081 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     4141 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     4201 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     4261 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     4321 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     4381 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     4441 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     4501 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     4561 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     4621 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     4681 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     4741 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     4801 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     4861 gctagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4921 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     4981 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     5041 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca
     5101 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     5161 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     5221 ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca
     5281 gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc
     5341 ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc
     5401 attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca
     5461 tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt
     5521 ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     5581 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     5641 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
     5701 atgagctgtt gacaattaat catcggctcg tataatgtgt ggaa
//
