LOCUS       sequence-175735-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of PTBP1-RRM12 C250S,C251S in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  AUTHORS   Dorn G, Leitner A, Boudet J, Campagne S, von Schroetter C, Moursy A,
            Aebersold R, Allain FH
  TITLE     Structural modeling of protein-RNA complexes using crosslinking of 
            segmentally isotope-labeled RNA and MS/MS.
  JOURNAL   Nat Methods. 2017 Mar 27. doi: 10.1038/nmeth.4235.
  PUBMED    28346450
REFERENCE   2  (bases 1 to 6126)
  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"; journalName: "Nat 
            Methods. 2017 Mar 27. doi: 10.1038/nmeth.4235."
COMMENT     Sequence Label: pET28a-PTBP1-RRM12CCtoSS
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     499..518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     752..769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    784..923
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(849..868)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1009..1031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1025..1216)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2025..3107)
                     /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     3069..3088
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3108..3185)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3391..3410
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3494..3513
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3494..3512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3513..3537
                     /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)."
     RBS             3552..3574
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3593..3610
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3620..3637
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4484..4501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4554..4572)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4568..4615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4652..5107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4739..4758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4949..4970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5199..6014)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5199..6014)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5922..5941
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6006..6025
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
       61 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
      121 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
      181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
      241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
      301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      661 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      721 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      781 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
      841 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
      901 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
      961 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1021 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     1081 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     1141 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     1201 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     1261 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     1321 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     1381 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     1441 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     1501 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     1561 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     1621 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     1681 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     1741 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     1801 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     1861 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     1921 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     1981 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     2041 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2101 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     2161 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     2221 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     2281 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     2341 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     2401 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     2461 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     2521 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2581 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2641 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2701 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2761 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2821 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2881 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2941 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     3001 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     3061 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     3121 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     3181 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     3241 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     3301 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     3361 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     3421 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     3481 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     3541 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
     3601 tcatcatcac agcagcggcc tggtgccgcg cggcagccat atgaacggaa atgacagcaa
     3661 gaagttcaaa ggtgacagcc gaagtgcagg cgtcccctct agagtgatcc acatccggaa
     3721 gctccccatc gacgtcacgg agggggaagt catctccctg gggctgccct ttgggaaggt
     3781 caccaacctc ctgatgctga aggggaaaaa ccaggccttc atcgagatga acacggagga
     3841 ggctgccaac accatggtga actactacac ctcggtgacc cctgtgctgc gcggccagcc
     3901 catctacatc cagttctcta accacaagga gctgaagacc gacagctctc ccaaccaggc
     3961 gcgggcccag gcggccctgc aggcggtgaa ctcggtccag tcggggaacc tggccttggc
     4021 tgcctcggcg gcggccgtgg acgcagggat ggcgatggcc gggcagagcc ctgtgctcag
     4081 gatcatcgtg gagaacctct tctaccctgt gaccctggat gtgctgcacc agattttctc
     4141 caagttcggc acagtgttga agatcatcac cttcaccaag aacaaccagt tccaggccct
     4201 gctgcagtat gcggaccccg tgagcgccca gcacgccaag ctgtcgctgg acgggcagaa
     4261 catctacaac gccagcagca cgctgcgcat cgacttttcc aagctcacca gcctcaacgt
     4321 caagtacaac aatgacaaga gccgtgacta cacacgccca gacctgcctt ccggggacag
     4381 ccagccctcg ctggaccaga ccatggccgc ggccttcggc ctttccgttc cgaacgtcca
     4441 cggcgccctg gcccccctgg ccatcccctc ggcgtgactc gagcaccacc accaccacca
     4501 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     4561 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     4621 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4681 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4741 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4801 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4861 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4921 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4981 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5041 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5101 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5161 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     5221 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     5281 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     5341 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     5401 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     5461 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     5521 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     5581 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     5641 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     5701 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     5761 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     5821 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     5881 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     5941 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     6001 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     6061 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6121 gatcaa
//
