LOCUS       sequence-176057-        6925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant expression of PTBP1 C250S,C251S in E.coli .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6925)
  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 6925)
  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-PTBP1CCtoSS
FEATURES             Location/Qualifiers
     source          1..6925
                     /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             5283..5300
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5353..5371)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5367..5414
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5451..5906
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5538..5557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5748..5769
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5998..6813)
                     /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(5998..6813)
                     /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     6721..6740
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6805..6824
                     /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 atggacggca ttgtcccaga
     3661 tatagccgtt ggtacaaagc ggggatctga cgagcttttc tctacttgtg tcactaacgg
     3721 accgtttatc atgagcagca actcggcttc tgcagcaaac ggaaatgaca gcaagaagtt
     3781 caaaggtgac agccgaagtg caggcgtccc ctctagagtg atccacatcc ggaagctccc
     3841 catcgacgtc acggaggggg aagtcatctc cctggggctg ccctttggga aggtcaccaa
     3901 cctcctgatg ctgaagggga aaaaccaggc cttcatcgag atgaacacgg aggaggctgc
     3961 caacaccatg gtgaactact acacctcggt gacccctgtg ctgcgcggcc agcccatcta
     4021 catccagttc tctaaccaca aggagctgaa gaccgacagc tctcccaacc aggcgcgggc
     4081 ccaggcggcc ctgcaggcgg tgaactcggt ccagtcgggg aacctggcct tggctgcctc
     4141 ggcggcggcc gtggacgcag ggatggcgat ggccgggcag agccctgtgc tcaggatcat
     4201 cgtggagaac ctcttctacc ctgtgaccct ggatgtgctg caccagattt tctccaagtt
     4261 cggcacagtg ttgaagatca tcaccttcac caagaacaac cagttccagg ccctgctgca
     4321 gtatgcggac cccgtgagcg cccagcacgc caagctgtcg ctggacgggc agaacatcta
     4381 caacgcctcc tccacgctgc gcatcgactt ttccaagctc accagcctca acgtcaagta
     4441 caacaatgac aagagccgtg actacacacg cccagacctg ccttccgggg acagccagcc
     4501 ctcgctggac cagaccatgg ccgcggcctt cggcctttcc gttccgaacg tccacggcgc
     4561 cctggccccc ctggccatcc cctcggcggc ggcggcagct gcggcggcag gtcggatcgc
     4621 catcccgggc ctggcggggg caggaaattc tgtattgctg gtcagcaacc tcaacccaga
     4681 gagagtcaca ccccaaagcc tctttattct tttcggcgtc tacggtgacg tgcagcgcgt
     4741 gaagatcctg ttcaataaga aggagaacgc cctagtgcag atggcggacg gcaaccaggc
     4801 ccagctggcc atgagccacc tgaacgggca caagctgcac gggaagccga tccgcatcac
     4861 gctctcgaag caccagaacg tgcagctgcc ccgcgagggc caggaggacc agggcctgac
     4921 caaggactac ggcaactcac ccctgcaccg cttcaagaag ccgggctcca agaacttcca
     4981 gaacatattc ccgccctcgg ccactctgca cctctccaac atcccgccct cagtctccga
     5041 ggaggatctc aaggtcctgt tttccagcaa tgggggcgtc gtcaaaggat tcaagttctt
     5101 ccagaaggac cgcaagatgg cactgatcca gatgggctcc gtggaggagg cggtccaggc
     5161 cctcattgac ctgcacaacc acgacctcgg ggagaaccac cacctgcggg tctccttctc
     5221 caagtccacc atctagggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg
     5281 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     5341 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     5401 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     5461 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5521 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5581 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5641 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5701 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5761 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5821 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5881 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     6001 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     6061 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     6121 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     6181 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     6241 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     6301 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     6361 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     6421 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     6481 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     6541 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     6601 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     6661 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     6721 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     6781 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     6841 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6901 cgtcagaccc cgtagaaaag atcaa
//
