LOCUS       sequence-476292-        7706 bp    DNA     circular SYN 18-JUN-2025
DEFINITION  Used for overexpression of human RUNX1 in mammalian cells. Contains 
            RUNX1 phosphomimetic mutations..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7706)
  AUTHORS   Kwon N, Lu YC, Thompson EN, Mancuso RI, Wang L, Zhang PX, Krause DS
  TITLE     CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in 
            megakaryocytic-erythroid progenitors.
  JOURNAL   Blood. 2024 Oct 24;144(17):1800-1812. doi: 10.1182/blood.2024023963.
  PUBMED    39102635
REFERENCE   2  (bases 1 to 7706)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1182/blood"; 
            journalName: "Blood"; date: "2024-10-24- 24"; volume: "144"; issue: 
            "17"; pages: "1800-1812"
COMMENT     Sequence Label: pMSCV-RUNX1-4D
FEATURES             Location/Qualifiers
     source          1..7706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1418..1442
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2841..2865)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        2880..3379
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2915..2938)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3312..3331
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3400..3999
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3400..3419)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3896..3916
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4099..4613
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4775..4791
                     /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)."
     promoter        complement(join(4799..4805,4806..4823,4824..4829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4844..4865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4982..4999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5153..5741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5233..5252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5912..6703,6704..6772))
                     /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     6535..6554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6773..6877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6945..6963
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7001..7023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7123..7142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7336..7358
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7486..7505
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7705..7706,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcatcaca agtttgtaca aaaaagcagg
     1441 cttcaccatg agagaatgca tacttggaat gaatccttct agagacgtcc acgatgccag
     1501 cacgagccgc cgcttcacgc cgccttccac cgcgctgagc ccaggcaaga tgagcgaggc
     1561 gttgccgctg ggcgccccgg acgccggcgc tgccctggcc ggcaagctga ggagcggcga
     1621 ccgcagcatg gtggaggtgc tggccgacca cccgggcgag ctggtgcgca ccgacagccc
     1681 caacttcctc tgctccgtgc tgcctacgca ctggcgctgc aacaagaccc tgcccatcgc
     1741 tttcaaggtg gtggccctag gggatgttcc agatggcact ctggtcactg tgatggctgg
     1801 caatgatgaa aactactcgg ctgagctgag aaatgctacc gcagccatga agaaccaggt
     1861 tgcaagattt aatgacctca ggtttgtcgg tcgaagtgga agagggaaaa gcttcactct
     1921 gaccatcact gtcttcacaa acccaccgca agtcgccacc taccacagag ccatcaaaat
     1981 cacagtggat gggccccgag aacctcgaag acatcggcag aaactagatg atcggaccaa
     2041 gcccgggagc ttgtcctttt ccgagcggct cagtgaactg gagcagctgc ggcgcacagc
     2101 catgagggtc agcccacacc acccagcccc cacgcccaac cctcgtgcct ccctgaacca
     2161 ctccactgcc tttaaccctc agcctcagag tcagatgcag gatacaaggc agatccaacc
     2221 agacccaccg tggtcctacg atcagtccta ccaatacctg ggatccattg ccgatccttc
     2281 tgtgcaccca gcagatccca ttgaccctgg acgtgccagc ggcatgacaa ccctctccgc
     2341 agaactttcc agtcgactct caacggcacc cgacctgaca gcgttcagcg acccgcgcca
     2401 gttccccgcg ctgccctcca tctccgaccc ccgcatgcac tatccaggcg ccttcaccta
     2461 ctccccgacg ccggtcacct cgggcatcgg catcggcatg tcggccatgg gctcggccac
     2521 gcgctaccac acctacctgc cgccgcccta ccccggctcg tcgcaagcgc agggaggccc
     2581 gttccaagcc agctcgccct cctaccacct gtactacggc gcctcggccg gctcctacca
     2641 gttctccatg gtgggcggcg agcgctcgcc gccgcgcatc ctgccgccct gcaccaacgc
     2701 ctccaccggc tccgcgctgc tcaaccccag cctcccgaac cagagcgacg tggtggaggc
     2761 cgagggcagc cacagcaact cccccaccaa catggcgccc tccgcgcgcc tggaggaggc
     2821 cgtgtggagg ccctactgag acccagcttt cttgtacaaa gtggtgatcg cgttctaccg
     2881 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     2941 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     3001 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     3061 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     3121 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     3181 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     3241 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     3301 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     3361 ctcatctccg ggcctttcga cctgcagccc aagcttacca tgaccgagta caagcccacg
     3421 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     3481 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3541 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3601 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3661 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3721 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3781 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3841 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3901 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3961 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     4021 cgcccgaccg aaaggagcgc acgaccccat gcatcgataa aataaaagat tttatttagt
     4081 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
     4141 acgccatttt gcaaggcatg gaaaatacat aactgagaat agagaagttc agatcaaggt
     4201 taggaacaga gagacagcag aatatgggcc aaacaggata tctgtggtaa gcagttcctg
     4261 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tcccgccctc agcagtttct
     4321 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
     4381 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
     4441 ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgatagact gcgtcgcccg
     4501 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
     4561 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catgggtaac
     4621 agtttcttga agttggagaa caacattctg agggtaggag tcgaatatta agtaatcctg
     4681 actcaattag ccactgtttt gaatccacat actccaatac tcctgaaata gttcattatg
     4741 gacagcgcag aaagagctgg ggagaattgt gaaattgtta tccgctcaca attccacaca
     4801 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4861 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4921 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4981 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5041 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5101 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5161 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5221 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5281 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5341 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5401 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5461 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5521 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5581 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5641 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5701 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5761 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5821 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5881 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5941 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6001 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6061 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6121 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6181 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6241 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6301 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6361 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6421 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6481 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6541 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6601 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6661 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6721 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6781 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6841 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6901 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     6961 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     7021 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     7081 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     7141 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     7201 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     7261 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     7321 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga atggtgcatg
     7381 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7441 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7501 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7561 gaggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac
     7621 tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat aaaagatttt
     7681 atttagtctc cagaaaaagg ggggaa
//
