LOCUS       sequence-201657-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express MBP hnRNPA2 LC with 4 R to K mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Ryan VH, Dignon GL, Zerze GH, Chabata CV, Silva R, Conicella AE, 
            Amaya J, Burke KA, Mittal J, Fawzi NL
  TITLE     Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, 
            Interactions, and Phase Separation Altered by Mutation and Arginine 
            Methylation.
  JOURNAL   Mol Cell. 2018 Jan 17. pii: S1097-2765(17)30979-6. doi: 
            10.1016/j.molcel.2017.12.022.
  PUBMED    29358076
REFERENCE   2  (bases 1 to 6912)
  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: "Mol Cell. 
            2018 Jan 17. pii: S1097-2765(17)30979-6. doi: 
            10.1016/j.molcel.2017.12.022."
COMMENT     Sequence Label: MBP-hnRNPA2_LC_R191K_R201K_R216K_R254K
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     937..956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(976..1053)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1259..1278
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1362..1381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1362..1380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1381..1405
                     /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             1420..1442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1467..1484
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1497..2594
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2451..2460
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2568..2591
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    2613..2637
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2646..2666
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3138..3155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3208..3226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3222..3269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3306..3761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3393..3412)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3603..3624
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3853..4668)
                     /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(3853..4668)
                     /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     4576..4595
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4660..4679
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4790..5378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5279..5298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5532..5549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5564..5703
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5629..5648)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5789..5811
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5805..5996)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6805..6912,1..975))
                     /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"
ORIGIN
        1 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
       61 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      121 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
      181 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      241 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      301 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
      361 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
      421 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
      481 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
      541 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
      601 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
      661 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
      721 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
      781 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
      841 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
      901 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
      961 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1021 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1081 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     1141 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     1201 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     1261 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     1321 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     1381 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     1441 gatataccat gggatctgat aaaattcatc atcatcatca tcacagcagc ggtaccaaaa
     1501 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
     1561 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
     1621 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
     1681 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
     1741 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
     1801 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
     1861 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
     1921 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
     1981 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
     2041 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
     2101 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
     2161 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
     2221 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
     2281 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
     2341 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
     2401 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccaccatgg
     2461 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     2521 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     2581 aagacgcgca gactaattcg ggatctgata tcacaagttt gtacaaaaaa gctgaaggcg
     2641 gtaccgagaa cctgtacttc cagggtcata tgggtaaagg tggcaacttt ggtttcggtg
     2701 atagcaaggg tggtggcggc aacttcggtc cgggtccggg cagcaatttc aagggtggtt
     2761 ccgacggtta cggtagcggt cgcggctttg gcgatggcta taatggttac ggtggtggtc
     2821 cgggcggcgg caatttcggt ggtagcccgg gttacggcgg tggtaaaggc ggttatggcg
     2881 gtggcggtcc aggctacggt aaccagggtg gtggttatgg tggtggctac gataactatg
     2941 gcggtggtaa ctatggtagc ggcaattaca atgattttgg caactacaac cagcaaccga
     3001 gcaattacgg cccgatgaaa tccggcaatt ttggcggttc ccgtaatatg ggtggtccgt
     3061 atggtggcgg taactacggc ccgggtggta gcggtggttc tggcggctat ggtggccgca
     3121 gccgttacta actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     3181 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3241 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     3301 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3361 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3421 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3481 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3541 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3601 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3661 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3721 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3781 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3841 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3901 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3961 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     4021 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     4081 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     4141 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     4201 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     4261 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     4321 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     4381 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4441 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4501 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4561 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4621 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     4681 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     4741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgatatcctt
     4801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4921 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5401 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5461 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5521 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5581 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     5641 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5701 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5761 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5821 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5881 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5941 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     6001 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     6061 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     6121 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     6181 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     6241 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     6301 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     6361 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     6421 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     6481 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     6541 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     6601 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     6661 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     6721 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     6781 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6841 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     6901 ggtggttttt ct
//
