LOCUS       sequence-175723-        7789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  recombinant expression of PTBP1-RRM2 C250S,C251S in e.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7789)
  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 7789)
  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: pTYB11-PTBP1-RRM2CCtoSS
FEATURES             Location/Qualifiers
     source          1..7789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      33..621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     522..541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     775..792
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(814..833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     974..996
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(990..1181)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1738..2820)
                     /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     2782..2801
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2821..2898)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3104..3123
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3207..3226
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3207..3225
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3226..3250
                     /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             3265..3287
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3340..4158
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             4165..4317
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
                     ALWQLQ"
     CDS             4321..4869
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="modified 3' region of the intein from the yeast 
                     Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 3' region
                     /note="C-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="GHGGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRE
                     TFLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT
                     KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI
                     TLSDDSDHQFLLGSQVVVQN"
     primer_bind     complement(5366..5384)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5380..5427
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5615..5632)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5615..5631)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5624..5646)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5810..5827)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5980..6066
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6158..6185
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        6204..6295
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6296..6364,6365..7156)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6514..6533)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7198..7711)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7393..7414)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7605..7624
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
       61 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
      121 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
      181 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
      241 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
      301 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
      361 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
      421 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
      481 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
      541 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      601 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      661 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
      721 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
      781 tcagtgagcg aggaagctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      841 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
      901 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      961 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     1021 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca gatgtctgcc
     1081 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     1141 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     1201 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     1261 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     1321 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagccgaa cgccagcaag
     1381 acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     1441 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     1501 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     1561 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     1621 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     1681 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1741 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1801 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1861 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1921 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1981 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     2041 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     2101 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     2161 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2221 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2281 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2341 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2401 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2461 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2521 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2581 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2641 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2701 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2761 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2821 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2881 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     2941 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     3001 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     3061 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     3121 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     3181 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     3241 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acttatgaaa
     3301 atcgaagaag gtaaactggt aatcggttcc ctggagggtt gctttgccaa gggtaccaat
     3361 gttttaatgg cggatgggtc tattgaatgt attgaaaaca ttgaggttgg taataaggtc
     3421 atgggtaaag atggcagacc tcgtgaggta attaaattgc ccagaggaag agaaactatg
     3481 tacagcgtcg tgcagaaaag tcagcacaga gcccacaaaa gtgactcaag tcgtgaagtg
     3541 ccagaattac tcaagtttac gtgtaatgcg acccatgagt tggttgttag aacacctcgt
     3601 agtgtccgcc gtttgtctcg taccattaag ggtgtcgaat attttgaagt tattactttt
     3661 gagatgggcc aaaagaaagc ccccgacggt agaattgttg agcttgtcaa ggaagtttca
     3721 aagagctacc caatatctga ggggcctgag agagccaacg aattagtaga atcctataga
     3781 aaggcttcaa ataaagctta ttttgagtgg actattgagg ccagagatct ttctctgttg
     3841 ggttcccatg ttcgtaaagc tacctaccag acttacgctc caattcttta tgagaatgac
     3901 cactttttcg actacatgca aaaaagtaag tttcatctca ccattgaagg tccaaaagta
     3961 cttgcttatt tacttggttt atggattggt gatggattgt ctgacagggc aactttttcg
     4021 gttgattcca gagatacttc tttgatggaa cgtgttactg aatatgctga aaagttgaat
     4081 ttgtgcgccg agtataagga cagaaaagaa ccacaagttg ccaaaactgt taatttgtac
     4141 tctaaagttg tcagaggtgc tagcacaaat cctggtgtat ccgcttggca ggtcaacaca
     4201 gcttatactg cgggacaatt ggtcacatat aacggcaaga cgtataaatg tttgcagccc
     4261 cacacctcct tggcaggatg ggaaccatcc aacgttcctg ccttgtggca gcttcaaggt
     4321 ggccatggtg gtattcgcaa taatcttaat actgagaatc cattatggga cgctattgtt
     4381 ggcttaggat tcttgaagga cggtgtcaaa aatattcctt ctttcttgtc tacggacaat
     4441 atcggtactc gtgaaacatt tcttgctggt ctaattgatt ctgatggcta tgttactgat
     4501 gagcatggta ttaaagcaac aataaagaca attcatactt ctgtcagaga tggtttggtt
     4561 tcccttgctc gttctttagg cttagtagtc tcggttaacg cagaacctgc taaggttgac
     4621 atgaatgtca ccaaacataa aattagttat gctatttata tgtctggtgg agatgttttg
     4681 cttaacgttc tttcgaagtg tgccggctct aaaaaattca ggcctgctcc cgccgctgct
     4741 tttgcacgtg agtgccgcgg attttatttc gagttacaag aattgaagga agacgattat
     4801 tatgggatta ctttatctga tgattctgat catcagtttt tgcttggatc ccaggttgtt
     4861 gtacagaacg ccgggcagag ccctgtgctc aggatcatcg tggagaacct cttctaccct
     4921 gtgaccctgg atgtgctgca ccagattttc tccaagttcg gcacagtgtt gaagatcatc
     4981 accttcacca agaacaacca gttccaggcc ctgctgcagt atgcggaccc cgtgagcgcc
     5041 cagcacgcca agctgtcgct ggacgggcag aacatctaca acgcctcctc cacgctgcgc
     5101 atcgactttt ccaagctcac cagcctcaac gtcaagtaca acaatgacaa gagccgtgac
     5161 tacacacgcc cagacctgcc ttccggggac agccagccct cgctggacca gaccatggcc
     5221 gcggccttcg gcctttccgt tccgaacgtc cacggcgccc tggcccccct ggccatcccc
     5281 tcggcgtgac tcgagcccgg gtgactgcag gaaggggatc cggctgctaa caaagcccga
     5341 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5401 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca
     5461 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     5521 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     5581 ttaactgtga taaactaccg cattaaagct tggcactggc cgtcgtttta caacgtcgtg
     5641 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     5701 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     5761 atggcgaatg gcagcttggc tgttttggcg gatgagataa gattttcagc ctgatacaga
     5821 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     5881 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     5941 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     6001 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     6061 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     6121 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     6181 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     6241 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6301 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6361 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6421 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6481 acgtttccca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     6541 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6601 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6661 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6721 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6781 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6841 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6901 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6961 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7021 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7081 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7141 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacc
     7201 ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt
     7261 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     7321 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     7381 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     7441 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     7501 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     7561 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     7621 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     7681 gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga
     7741 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcg
//
