LOCUS       sequence-273980-        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of PCBP4 with dual-affinity tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  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 6140)
  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: PCBP4_3xKH_pGEX
FEATURES             Location/Qualifiers
     source          1..6140
                     /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(1972..1994)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2139..2157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2225..2329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2330..2398,2399..3190)
                     /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(2548..2567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3361..3949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3850..3869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4103..4120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4193..4270
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4271..5353
                     /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(4290..4309)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5402..5407,5408..5425,5426..5432)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5440..5456
                     /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     5464..5480
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5464..5480
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5476..6003
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5496..5513)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5496..5512)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5505..5527)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6105..6110,6111..6126,6127..6133)
                     /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 ccatgagcgg ctcggacggg ggactggagg aggagccaga gctcagcatc
      901 accctcacgc tgcggatgct gatgcacggg aaggaagtgg gcagcatcat cgggaagaag
      961 ggcgagactg taaagcgaat ccgggagcag agcagtgccc ggatcaccat ctccgagggc
     1021 tcctgccctg aacgcatcac caccatcacc gggtctacag cagctgtctt ccatgcagtc
     1081 tccatgattg ctttcaaact ggatgaggac ctttgtgctg ctcctgcaaa tggtggaaat
     1141 gtctccaggc ctccagtgac cctgcgcctt gtcatccctg ccagtcagtg tggctcactg
     1201 attgggaagg ctggcaccaa gatcaaggag atccgagaga ctacgggtgc ccaggtacag
     1261 gtggcagggg acctgctccc caactccaca gagcgagctg ttacggtatc tggggtgcct
     1321 gatgccatca tcctgtgtgt gcgccagatc tgcgctgtta tcctggagtc cccacccaaa
     1381 ggagccacta tcccctacca tccgagcctc tccctaggta ctgttcttct ctctgccaac
     1441 cagggcttct ctgtccaggg tcagtatggg gctgtgaccc cagctgaggt caccaagctc
     1501 cagcagctct caagccatgc ggtccccttt gccacaccca gcgtggtgcc aggactggat
     1561 cccggcacac agaccagctc acaggagttc ttggttccca acgatttgat tggctgtgtg
     1621 atcgggcgcc agggcagcaa gatcagcgag atccggcaga tgtcaggggc acatatcaag
     1681 atcgggaacc aagcagaggg cgctggggag cggcatgtca ccatcactgg ctctccggtc
     1741 tccatcgccc tggcccagta cctcatcact gcctgtctag agacggccaa gtctacctct
     1801 ggggggacgc ccagctcggc ccccgcagac ctgcctgccc ccttctcgcc acccctgacg
     1861 gccctgccca cagctccccc tggcctgctg ggcacaccct atgccatctg agcggccgca
     1921 tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     1981 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2041 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     2101 tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt
     2161 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2221 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2281 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2341 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2401 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2461 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2521 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     2581 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2641 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2701 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2761 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2821 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2881 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2941 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3001 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3061 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3121 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3181 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3241 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3301 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3361 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3421 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3481 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3541 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3601 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3661 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3721 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3781 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3841 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3901 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3961 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4021 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4081 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4141 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat
     4201 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     4261 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     4321 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     4381 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     4441 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     4501 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     4561 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     4621 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     4681 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4741 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4801 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     4861 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     4921 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     4981 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     5041 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     5101 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     5161 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     5221 gctgttgccc gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc
     5281 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5341 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg
     5401 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5461 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
     5521 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5581 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5641 aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg
     5701 gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt
     5761 tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt
     5821 cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta
     5881 caggaaggcc agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg
     5941 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     6001 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     6061 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     6121 ggctcgtata atgtgtggaa
//
