LOCUS       sequence-311809-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of 6xHis RUNX2(+10A) IDR mCherry Fusion 
            Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  AUTHORS   Basu S, Mackowiak SD, Niskanen H, Knezevic D, Asimi V, Grosswendt S,
            Geertsema H, Ali S, Jerkovic I, Ewers H, Mundlos S, Meissner A, 
            Ibrahim DM, Hnisz D
  TITLE     Unblending of Transcriptional Condensates in Human Repeat Expansion 
            Disease.
  JOURNAL   Cell. 2020 May 28;181(5):1062-1079.e30. doi: 
            10.1016/j.cell.2020.04.018. Epub 2020 May 7.
  PUBMED    32386547
REFERENCE   2  (bases 1 to 6286)
  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"; doi: 
            "10.1016/j.cell.2020.04.018"; journalName: "Cell"; date: 
            "2020-05-28- 28"; volume: "181"; issue: "5"; pages: "1062-1079.e30"
COMMENT     Sequence Label: pET-45b-RUNX2(+10A)-IDR-mCherry
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             36..53
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      84..93
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1194..1238
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1276..1294)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1290..1337
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1374..1829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1461..1480)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1671..1692
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1944..2735,2736..2804))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2567..2586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2805..2897)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2978..3566
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3467..3486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3720..3737
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3752..3891
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3817..3836)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3977..3999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3993..4184)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4756..5838)
                     /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     5800..5819
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5839..5916)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6122..6141
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6229..6248
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6229..6247
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6248..6272
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tggcacatca ccaccaccat cacgtgggta
       61 ccggtggaag agaggccact agtgccacca tggcatcaaa cagcctcttc agcacagtga
      121 caccatgtca gcaaaacttc ttttgggatc cgagcaccag ccggcgcttc agccccccct
      181 ccagcagcct gcagcccggc aaaatgagcg acgtgagccc ggtggtggct gcgcaacagc
      241 agcagcaaca gcagcagcag caacagcagc agcagcagca gcaacagcag cagcagcagc
      301 aggaggcggc ggcagccgcg gctgccgcag cggctgcagc ggcggcggct gcggcggcgg
      361 cggcggctgc ggcggcggca gctgcagtgc cccggttgcg gccgccccac gacaaccgca
      421 ccatggtgga gacgtacggg gtctggctcg tcgggtccat ggtttccaaa ggagaggagg
      481 ataatatggc tataattaaa gagtttatgc ggttcaaggt gcatatggag ggatctgtga
      541 acggtcacga gttcgaaatc gagggcgaag gcgaggggcg accctatgaa gggacacaga
      601 cggccaaact gaaggtgacc aagggtggcc ccctgccttt cgcctgggac atcctgtcac
      661 cccagtttat gtacggctct aaggcttacg ttaagcaccc tgccgatatt ccggactact
      721 tgaaactgag cttccctgaa gggttcaagt gggaacgggt aatgaatttt gaggatggcg
      781 gtgtagtcac agttacccag gacagctctc ttcaggacgg agaatttatc tataaggtta
      841 aacttcgggg cactaacttc ccatcagacg gccccgtgat gcagaagaaa actatgggct
      901 gggaagccag ctcagaacgc atgtatcccg aggacggagc actgaaggga gaaatcaagc
      961 agcgactcaa gctgaaggat gggggacatt atgatgctga ggtcaaaacc acctataagg
     1021 ccaagaaacc cgttcagctt cctggggcat acaacgtgaa tatcaaactg gatattacat
     1081 cccataacga agactatacc atcgtggaac agtacgagcg ggccgagggc agacatagca
     1141 cagggggcat ggatgaattg tacaagaagc ttgcggccgc actcgagtct ggtaaagaaa
     1201 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     1261 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1321 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1381 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1441 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1501 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1561 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1621 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1681 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1741 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1801 ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag attatcaaaa
     1861 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1921 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1981 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2041 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2101 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2161 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2221 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2281 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2341 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2401 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2461 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2521 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2581 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2641 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2701 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2761 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2821 tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2881 ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt tcgttccact
     2941 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3001 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3061 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3121 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3181 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3241 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3301 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3361 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3421 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3481 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3541 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3601 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3661 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3721 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3781 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3841 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3901 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3961 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4021 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4081 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4141 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4201 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4261 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4321 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4381 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4441 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4501 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4561 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4621 aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga gctgactggg
     4681 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4741 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4801 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4861 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4921 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4981 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     5041 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5101 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5161 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5221 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5281 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5341 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5401 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5461 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5521 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5581 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5641 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5701 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5761 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5821 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5881 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5941 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6001 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6061 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6121 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6181 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca
     6241 ctatagggga attgtgagcg gataacaatt cccctctaga aataat
//
