LOCUS       sequence-311806-        6256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of  6xHis RUNX2(WT) IDR mCherry Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  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 6256)
  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(WT)-IDR-mCherry
FEATURES             Location/Qualifiers
     source          1..6256
                     /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             1164..1208
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1246..1264)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1260..1307
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1344..1799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1431..1450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1641..1662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1914..2705,2706..2774))
                     /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     2537..2556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2775..2867)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2948..3536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3437..3456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3690..3707
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3722..3861
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3787..3806)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3947..3969
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3963..4154)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4726..5808)
                     /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     5770..5789
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5809..5886)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6092..6111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6199..6218
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6199..6217
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6218..6242
                     /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 ggcggccgct gcggcggcgg cggcggctgc ggcggcggca gctgcagtgc
      361 cccggttacg gccgccccac gacaaccgca ccatggtgga gacgtacggg gtctggctcg
      421 tcgggtccat ggtttccaaa ggagaggagg ataatatggc tataattaaa gagtttatgc
      481 ggttcaaggt gcatatggag ggatctgtga acggtcacga gttcgaaatc gagggcgaag
      541 gcgaggggcg accctatgaa gggacacaga cggccaaact gaaggtgacc aagggtggcc
      601 ccctgccttt cgcctgggac atcctgtcac cccagtttat gtacggctct aaggcttacg
      661 ttaagcaccc tgccgatatt ccggactact tgaaactgag cttccctgaa gggttcaagt
      721 gggaacgggt aatgaatttt gaggatggcg gtgtagtcac agttacccag gacagctctc
      781 ttcaggacgg agaatttatc tataaggtta aacttcgggg cactaacttc ccatcagacg
      841 gccccgtgat gcagaagaaa actatgggct gggaagccag ctcagaacgc atgtatcccg
      901 aggacggagc actgaaggga gaaatcaagc agcgactcaa gctgaaggat gggggacatt
      961 atgatgctga ggtcaaaacc acctataagg ccaagaaacc cgttcagctt cctggggcat
     1021 acaacgtgaa tatcaaactg gatattacat cccataacga agactatacc atcgtggaac
     1081 agtacgagcg ggccgagggc agacatagca cagggggcat ggatgaattg tacaagaagc
     1141 ttgcggccgc actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     1201 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     1261 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1321 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1381 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1441 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1501 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1561 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1621 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1681 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1741 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     1801 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1861 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1921 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1981 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2041 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     2101 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2161 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2221 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2281 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2341 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2401 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2461 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2521 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2581 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2641 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2701 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2761 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     2821 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     2881 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2941 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3001 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3061 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3121 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3181 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3241 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3301 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3361 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3421 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3481 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3541 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3601 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3661 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3721 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3781 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3841 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3901 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3961 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4021 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4081 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4141 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4201 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4261 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4321 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4381 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4441 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4501 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4561 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     4621 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4681 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4861 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4921 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4981 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     5041 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5101 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5161 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5221 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5281 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5341 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5401 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5461 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5521 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5581 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5641 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5701 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5761 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5821 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5881 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5941 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6001 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6061 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6121 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     6181 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     6241 cccctctaga aataat
//
