LOCUS       sequence-201658-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express MBP hnRNPA2 LC with 2 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_R254K
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             188..205
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(258..276)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      272..319
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      356..811
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(443..462)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     653..674
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(903..1718)
                     /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(903..1718)
                     /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     1626..1645
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1710..1729
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1840..2428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2329..2348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2582..2599
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2614..2753
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2679..2698)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2839..2861
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2855..3046)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3855..4937)
                     /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     4899..4918
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4938..5015)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5221..5240
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5324..5343
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5324..5342
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5343..5367
                     /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             5382..5404
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5429..5446
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5459..6556
                     /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      6413..6422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6530..6553
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    6575..6599
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6608..6628
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 ctatggtagc ggcaattaca atgattttgg caactacaac cagcaaccga gcaattacgg
       61 cccgatgaaa tccggcaatt ttggcggttc ccgtaatatg ggtggtccgt atggtggcgg
      121 taactacggc ccgggtggta gcggtggttc tggcggctat ggtggccgca gccgttacta
      181 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
      241 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      301 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      361 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      421 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      481 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      541 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
      601 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      661 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      721 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      781 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
      841 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
      901 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
      961 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1021 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1081 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1141 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1201 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1261 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1321 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     1381 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     1441 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     1501 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     1561 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     1621 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     1681 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     1741 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     1801 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgatatcctt tttttctgcg
     1861 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1921 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1981 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2041 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2101 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2161 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2221 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2281 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2341 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2401 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2461 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2521 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2581 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2641 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2701 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2761 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2821 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2881 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2941 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3001 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3061 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3121 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3181 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3241 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3301 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3361 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3421 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3481 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     3541 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3601 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3661 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3721 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3781 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3841 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3901 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3961 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4021 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4081 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4141 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4201 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4261 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4321 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4381 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4441 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4501 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4561 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4621 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4681 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4741 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4801 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4861 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4921 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4981 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5041 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5101 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5161 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     5221 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     5281 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     5341 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     5401 gagatatacc atgggatctg ataaaattca tcatcatcat catcacagca gcggtaccaa
     5461 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
     5521 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
     5581 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
     5641 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
     5701 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
     5761 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
     5821 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
     5881 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
     5941 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt
     6001 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
     6061 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
     6121 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
     6181 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
     6241 gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
     6301 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     6361 cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccaccat
     6421 ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta
     6481 tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct
     6541 gaaagacgcg cagactaatt cgggatctga tatcacaagt ttgtacaaaa aagctgaagg
     6601 cggtaccgag aacctgtact tccagggtca tatgggtaaa ggtggcaact ttggtttcgg
     6661 tgatagccgc ggtggtggcg gcaacttcgg tccgggtccg ggcagcaatt tccgtggtgg
     6721 ttccgacggt tacggtagcg gtcgcggctt tggcgatggc tataatggtt acggtggtgg
     6781 tccgggcggc ggcaatttcg gtggtagccc gggttacggc ggtggtaaag gcggttatgg
     6841 cggtggcggt ccaggctacg gtaaccaggg tggtggttat ggtggtggct acgataacta
     6901 tggcggtggt aa
//
