LOCUS       sequence-382280-        8729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of mCherry fused to human Gata2 with 
            mutation R307W in mammalian cells .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8729)
  AUTHORS   Silverio-Alves R, Kurochkin I, Rydstrom A, Vazquez Echegaray C, 
            Haider J, Nicholls M, Rode C, Thelaus L, Lindgren AY, Ferreira AG, 
            Brandao R, Larsson J, de Bruijn MFTR, Martin-Gonzalez J, Pereira CF
  TITLE     GATA2 mitotic bookmarking is required for definitive haematopoiesis.
  JOURNAL   Nat Commun. 2023 Aug 14;14(1):4645. doi: 10.1038/s41467-023-40391-x.
  PUBMED    37580379
REFERENCE   2  (bases 1 to 8729)
  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: "Nat 
            Commun."; date: "2023-08-14"; volume: "14(1):4645. doi"; pages: " 
            10.1038/s41467-023-40391-x"
COMMENT     Sequence Label: SFFV-mCh-GATA2-R307W
FEATURES             Location/Qualifiers
     source          1..8729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      31..486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(118..137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     328..349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        512..616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(617..685,686..1477)
                     /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     complement(835..854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1648..2236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2137..2156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2390..2407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2524..2545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2560..2565,2566..2583,2584..2590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2598..2614
                     /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)."
     primer_bind     2603..2625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2622..2638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2622..2638
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2657..2677
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2659..2677
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2705..2931
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2932..3112
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3159..3284
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3777..4010
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4195..4239
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    4506..4623
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4657..5063
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             5098..5799
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(5231..5249)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5499..5518
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5693..5716
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    7456..7859
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             7952..8185
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8257..8378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8294..8313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8348..8367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8418..8553
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8573..8592)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8574..8592)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8602..8619)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8602..8618)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8611..8633)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
       61 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      121 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      181 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      241 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      301 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      361 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      421 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
      481 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
      541 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      601 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      661 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      721 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      781 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      841 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      901 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      961 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1021 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1081 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1141 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     1201 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1261 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     1321 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     1381 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     1441 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1501 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1561 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     1621 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1681 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1741 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1801 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1861 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1921 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1981 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2041 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2101 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2161 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2221 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     2281 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2341 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2401 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2461 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2521 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     2581 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     2641 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
     2701 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
     2761 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
     2821 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
     2881 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
     2941 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     3001 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     3061 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
     3121 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
     3181 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
     3241 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
     3301 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
     3361 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     3421 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     3481 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     3541 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     3601 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     3661 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     3721 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     3781 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     3841 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     3901 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     3961 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     4021 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     4081 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     4141 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     4201 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     4261 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     4321 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     4381 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     4441 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcgg
     4501 ttaactttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     4561 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     4621 tttatcgatc acgagactag cctcgagctg gctgcagtaa cgccattttg caaggcatgg
     4681 aaaaatacca aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta
     4741 acgttgggcc aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa
     4801 cagatggtca ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat
     4861 ggcccaaccc tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc
     4921 tgaaatgacc ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc
     4981 gcgcttctgc ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc
     5041 ctccgacaga ctgagtcggc cggtcgaatc aagcttatcg ataccgtcga cggatccatg
     5101 gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg catggagggc
     5161 tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     5221 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
     5281 ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc
     5341 gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     5401 gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac
     5461 aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc
     5521 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag
     5581 atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagaccatc
     5641 tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac
     5701 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc
     5761 caccacctgt tcctgtacgg catggacgag ctgtacaagg aggtggcgcc cgagcagccg
     5821 cgctggatgg cgcacccggc cgtgctgaat gcgcagcacc ccgactcaca ccacccgggc
     5881 ctggcgcaca actacatgga acccgcgcag ctgctgcctc cagacgaggt ggacgtcttc
     5941 ttcaatcacc tcgactcgca gggcaacccc tactatgcca accccgctca cgcgcgggcg
     6001 cgcgtctcct acagccccgc gcacgcccgc ctgaccggag gccagatgtg ccgcccacac
     6061 ttgttgcaca gcccgggttt gccctggctg gacgggggca aagcagccct ctctgccgct
     6121 gcggcccacc accacaaccc ctggaccgtg agccccttct ccaagacgcc actgcacccc
     6181 tcagctgctg gaggccctgg aggcccactc tctgtgtacc caggggctgg gggtgggagc
     6241 gggggaggca gcgggagctc agtggcctcc ctcaccccta cagcagccca ctctggctcc
     6301 caccttttcg gcttcccacc cacgccaccc aaagaagtgt ctcctgaccc tagcaccacg
     6361 ggggctgcgt ctccagcctc atcttccgcg gggggtagtg cagcccgagg agaggacaag
     6421 gacggcgtca agtaccaggt gtcactgacg gagagcatga agatggaaag tggcagtccc
     6481 ctgcgcccag gcctagctac tatgggcacc cagcctgcta cacaccaccc catccccacc
     6541 tacccctcct atgtgccggc ggctgcccac gactacagca gcggactctt ccaccccgga
     6601 ggcttcctgg ggggaccggc ctccagcttc acccctaagc agcgcagcaa ggctcgttcc
     6661 tgttcagaag gccgggagtg tgtcaactgt ggggccacag ccacccctct ctggtggcgg
     6721 gacggcaccg gccactacct gtgcaatgcc tgtggcctct accacaagat gaatgggcag
     6781 aaccgaccac tcatcaagcc caagcgaaga ctgtcggccg ccagaagagc cggcacctgt
     6841 tgtgcaaatt gtcagacgac aaccaccacc ttatggcgcc gaaacgccaa cggggaccct
     6901 gtctgcaacg cctgtggcct ctactacaag ctgcacaatg ttaacaggcc actgaccatg
     6961 aagaaggaag ggatccagac tcggaaccgg aagatgtcca acaagtccaa gaagagcaag
     7021 aaaggggcgg agtgcttcga ggagctgtca aagtgcatgc aggagaagtc atcccccttc
     7081 agtgcagctg ccctggctgg acacatggca cctgtgggcc acctcccgcc cttcagccac
     7141 tccggacaca tcctgcccac tccgacgccc atccacccct cctccagcct ctccttcggc
     7201 cacccccacc cgtccagcat ggtgaccgcc atgggctagg gatcccccgg gctgcaggaa
     7261 ttcgagcatc ttaccgccat ttattcccat atttgttctg tttttcttga tttgggtata
     7321 catttaaatg ttaataaaac aaaatggtgg ggcaatcatt tacattttta gggatatgta
     7381 attactagtt caggtgtatt gccacaagac aaacatgtta agaaactttc ccgttattta
     7441 cgctctgttc ctgttaatca acctctggat tacaaaattt gtgaaagatt gactgatatt
     7501 cttaactatg ttgctccttt tacgctgtgt ggatatgctg ctttaatgcc tctgtatcat
     7561 gctattgctt cccgtacggc tttcgttttc tcctccttgt ataaatcctg gttgctgtct
     7621 ctttatgagg agttgtggcc cgttgtccgt caacgtggcg tggtgtgctc tgtgtttgct
     7681 gacgcaaccc ccactggctg gggcattgcc accacctgtc aactcctttc tgggactttc
     7741 gctttccccc tcccgatcgc cacggcagaa ctcatcgccg cctgccttgc ccgctgctgg
     7801 acaggggcta ggttgctggg cactgataat tccgtggtgt tgtcggggaa gctgacgtcc
     7861 tttgcaattc gatatcaagc tgtaccttta agaccaatga cttacaaggc agctgtagat
     7921 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     7981 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     8041 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     8101 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     8161 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     8221 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     8281 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     8341 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     8401 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     8461 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     8521 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     8581 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     8641 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     8701 cgaagaggcc cgcaccgatc gcccttccc
//
