LOCUS       sequence-273985-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of PUM1 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  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 6320)
  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: PUM1_PUM_pGEX
FEATURES             Location/Qualifiers
     source          1..6320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             769..882
                     /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(2188..2210)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2355..2373)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2441..2545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2546..2614,2615..3406)
                     /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(2764..2783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3577..4165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4066..4085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4319..4336
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4409..4486
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4487..5569
                     /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(4506..4525)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5618..5623,5624..5641,5642..5648)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5656..5672
                     /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     5680..5696
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5680..5696
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5692..6219
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5712..5729)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5712..5728)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggattaat ggacgaaaaa
      781 accaccggtt ggcgtggtgg tcacgttgtt gaaggtctgg ctggtgaact ggaacagctg
      841 cgtgctcgtc tggaacacca cccgcagggt cagcgtgaac cgggatccgg aggactcacg
      901 aacggcagtg gaagatacat ctctgctgct ccaggcgctg aagccaagta ccgcagtgca
      961 agcagcgcct ccagcctctt cagcccgagc agcactcttt tctcttcctc tcgtttgcga
     1021 tatggaatgt ctgatgtcat gccttctggc aggagcaggc ttttggaaga ttttcgaaac
     1081 aaccggtacc ccaatttaca actgcgggag attgctggac atataatgga attttcccaa
     1141 gaccagcatg ggtccagatt cattcagctg aaactggagc gtgccacacc agctgagcgc
     1201 cagcttgtct tcaatgaaat cctccaggct gcctaccaac tcatggtgga tgtgtttggt
     1261 aattacgtca ttcagaagtt ctttgaattt ggcagtcttg aacagaagct ggctttggca
     1321 gaacggattc gaggccacgt cctgtcattg gcactacaga tgtatggctg ccgtgttatc
     1381 cagaaagctc ttgagtttat tccttcagac cagcagaatg agatggttcg ggaactagat
     1441 ggccatgtct tgaagtgtgt gaaagatcag aatggcaatc acgtggttca gaaatgcatt
     1501 gaatgtgtac agccccagtc tttgcaattt atcatcgatg cgtttaaggg acaggtattt
     1561 gccttatcca cacatcctta tggctgccga gtgattcaga gaatcctgga gcactgtctc
     1621 cctgaccaga cactccctat tttagaggag cttcaccagc acacagagca gcttgtacag
     1681 gatcaatatg gaaattatgt aatccaacat gtactggagc acggtcgtcc tgaggataaa
     1741 agcaaaattg tagcagaaat ccgaggcaat gtacttgtat tgagtcagca caaatttgca
     1801 agcaatgttg tggagaagtg tgttactcac gcctcacgta cggagcgcgc tgtgctcatc
     1861 gatgaggtgt gcaccatgaa cgacggtccc cacagtgcct tatacaccat gatgaaggac
     1921 cagtatgcca actacgtggt ccagaagatg attgacgtgg cggagccagg ccagcggaag
     1981 atcgtcatgc ataagatccg gccccacatc gcaactcttc gtaagtacac ctatggcaag
     2041 cacattctgg ccaagctgga gaagtactac atgaagaacg gtgttgactt agggcccatc
     2101 tgtggccccc ctaatggtat catctgagcg gccgcatcgt gactgactga cgatctgcct
     2161 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2221 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2281 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     2341 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2401 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2761 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     2821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3061 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3241 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3721 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4201 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4261 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4321 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4381 gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg
     4441 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     4501 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     4561 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     4621 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     4681 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     4741 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4801 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4861 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4921 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     4981 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     5041 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     5101 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     5161 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     5221 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     5281 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     5341 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     5401 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     5461 aagaaaaacc acgctagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     5521 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     5581 ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     5641 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     5701 attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5761 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5821 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg
     5881 tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat
     5941 actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac
     6001 gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt
     6061 tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt
     6121 tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc tggcgtcagg
     6181 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     6241 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
     6301 aaatattctg aaatgagctg
//
