LOCUS       sequence-274467-        5585 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recombinant expression of BOLL with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5585)
  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 5585)
  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"; 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: BOLL_RRM_pGEX
FEATURES             Location/Qualifiers
     source          1..5585
                     /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(1417..1439)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1584..1602)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1670..1774
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1775..1843,1844..2635)
                     /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(1993..2012)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2806..3394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3295..3314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3548..3565
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3638..3715
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3716..4798
                     /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(3735..3754)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4847..4852,4853..4870,4871..4877)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4885..4901
                     /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     4909..4925
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4909..4925
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4921..5448
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4941..4958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4941..4957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5550..5555,5556..5571,5572..5578)
                     /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 ccatgcaaac agattcatta tctccatccc ctaatcctgt gtcacctgtg
      901 cctttgaata acccaacaag tgccccaaga tatggaacag tgatccctaa tcgcatcttt
      961 gtaggaggaa ttgattttaa gacaaacgaa agtgatttaa gaaaattttt ttcccagtat
     1021 gggtctgtga aagaagtgaa gattgtaaat gacagagctg gagtatccaa agggtatggt
     1081 ttcgtcactt ttgaaacaca agaagatgca caaaaaattt tacaagaggc tgaaaaactt
     1141 aattataagg ataagaagct gaacattggt ccagcaataa gaaaacaaca agtagggatc
     1201 cctcgttcta gtataatgcc agcagctgga acaatgtatc taacaacttc aactggatat
     1261 ccttatactt accataatgg tgttgcttat tttcatactc cagaggtaac ttcggtccca
     1321 ccgccttggc cttcacgttc tgtatgtagc tcctgagcgg ccgcatcgtg actgactgac
     1381 gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     1441 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     1501 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     1561 gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1621 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1681 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1741 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1801 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1861 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1921 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1981 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     2041 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2101 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2161 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2221 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2281 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2341 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2401 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2461 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2521 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2581 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2641 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2701 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3481 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3541 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3601 tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat ggcgcaaaac
     3661 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     3721 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     3781 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     3841 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     3901 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     3961 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     4021 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     4081 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     4141 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     4201 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     4261 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     4321 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     4381 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     4441 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     4501 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     4561 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     4621 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     4681 actggtgaaa agaaaaacca cgctagcgcc caatacgcaa accgcctctc cccgcgcgtt
     4741 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4801 gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc
     4861 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     4921 atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4981 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     5041 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     5101 tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct
     5161 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc
     5221 tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg
     5281 acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg
     5341 cgaattattt ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct
     5401 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa
     5461 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
     5521 ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg
     5581 tggaa
//
