LOCUS       sequence-273979-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of RBFOX3 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  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 6671)
  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: RBFOX3_RRM_pGEX
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             23..676
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     634..656
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             683..706
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             716..829
                     /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             836..1726
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1739..1759
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2486..2508)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2653..2671)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2739..2843
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2844..2912,2913..3704)
                     /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(3062..3081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3875..4463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4364..4383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4617..4634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4707..4784
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4785..5867
                     /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(4804..4823)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5916..5921,5922..5939,5940..5946)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5954..5970
                     /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     5978..5994
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5978..5994
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5990..6517
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6010..6027)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6010..6026)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6019..6041)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6619..6624,6625..6640,6641..6647)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6655..6671
                     /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)."
ORIGIN
        1 tttcacacag gaaacagtat tcatgtcccc tatactaggt tattggaaaa ttaagggcct
       61 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
      121 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
      181 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
      241 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
      301 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
      361 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
      421 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
      481 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
      541 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
      601 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
      661 cgaccatcct ccaaaatcgg atctggaagt tctgttccag gggcccctgg gattaatgga
      721 cgaaaaaacc accggttggc gtggtggtca cgttgttgaa ggtctggctg gtgaactgga
      781 acagctgcgt gctcgtctgg aacaccaccc gcagggtcag cgtgaaccgg gatctatggc
      841 agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
      901 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
      961 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     1021 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     1081 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     1141 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     1201 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc
     1261 agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat
     1321 catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac
     1381 tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact
     1441 gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt
     1501 cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac
     1561 cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg
     1621 caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat
     1681 cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt tccggcgagc caaccactga
     1741 ggatctgtac tttcagagcg atacgcgtga accgggatcc acagagcatg gcatgaccct
     1801 gtacacacca gcacagaccc accccgagca gccaggctcc gaggccagca cacagcccat
     1861 cgccgggacc cagacagtgc cgcagacaga cgaggcggca cagacggaca gccagccgct
     1921 ccacccctcc gaccctacag agaagcagca gcccaagcgg ctacacgtct ccaacatccc
     1981 cttccggttc agggaccccg acttgcggca aatgttcggg caattcggaa aaattttaga
     2041 cgtggagatc atttttaacg agcggggctc caagggtttt gggtttgtaa cttttgaaac
     2101 tagctcagat gctgaccgag cccgggagaa gctgaatggg acgatcgtag agggacggaa
     2161 aattgaggtc aataatgcca cggcccgagt gatgaccaac aagaagacgg ggaaccccta
     2221 caccaacggc tggaagctaa atccagtggt cggcgcagtc tacgggcctg aattctatgc
     2281 agtgacgggg ttcccctacc ccaccaccgg cacagccgtt gcctaccggg gcgcacatct
     2341 tcggggccgg ggccgggccg tgtataatac atttcgggct gcgccacccc caccccccat
     2401 cccgacttac ggagcggtcg tgtgagcggc cgcatcgtga ctgactgacg atctgcctcg
     2461 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2521 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2581 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     2641 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2701 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2761 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2821 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2881 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2941 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3001 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3061 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3121 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3181 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3241 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3301 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3361 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3421 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3481 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3541 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3601 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3661 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3721 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3781 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3841 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3901 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3961 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4021 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4081 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4141 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4201 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4261 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4321 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4381 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4441 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4501 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4561 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4621 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4681 gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta
     4741 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4801 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4861 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4921 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4981 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5041 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5101 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5161 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5221 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5281 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5341 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5401 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5461 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5521 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5581 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5641 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5701 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5761 gaaaaaccac gctagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5821 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5881 aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5941 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6001 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6061 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6121 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     6181 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     6241 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     6301 gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     6361 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     6421 tgatggcgtt ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca
     6481 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
     6541 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
     6601 atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt ggaattgtga
     6661 gcggataaca a
//
