LOCUS       sequence-382294-        8729 bp    DNA     circular SYN 15-APR-2025
DEFINITION  constitutive expression of mCherry fused to human Gata2 with 
            mutation R398W 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 Apr 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-R398W
FEATURES             Location/Qualifiers
     source          1..8729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(161..180)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(162..180)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(190..207)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(190..206)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(199..221)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      348..803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(435..454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     645..666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        829..933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(934..1002,1003..1794)
                     /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(1152..1171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1965..2553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2454..2473
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2707..2724
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2841..2862
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2877..2882,2883..2900,2901..2907)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2915..2931
                     /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     2920..2942
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2939..2955
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2939..2955
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2974..2994
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2976..2994
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3022..3248
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3249..3429
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3476..3601
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4094..4327
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4512..4556
                     /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    4823..4940
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4974..5380
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             5415..6116
                     /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(5548..5566)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5816..5835
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6010..6033
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    7773..8176
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             8269..8502
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8574..8695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8611..8630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8665..8684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
       61 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
      121 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
      181 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
      241 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
      301 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
      361 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      421 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      481 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      541 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
      601 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      661 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
      721 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
      781 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
      841 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      901 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      961 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1021 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1081 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1141 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1201 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1261 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1321 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     1381 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     1441 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     1501 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     1561 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     1621 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     1681 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1741 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1801 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1861 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1921 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1981 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2041 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2101 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2161 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2221 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2281 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2341 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2401 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2461 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2521 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2581 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2641 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2701 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     2761 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     2821 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     2881 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     2941 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     3001 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
     3061 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
     3121 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
     3181 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
     3241 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     3301 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     3361 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     3421 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     3481 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     3541 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     3601 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     3661 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     3721 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     3781 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     3841 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     3901 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     3961 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     4021 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     4081 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     4141 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     4201 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     4261 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     4321 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     4381 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     4441 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     4501 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     4561 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     4621 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     4681 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     4741 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     4801 gatctcgacg gtatcggtta acttttaaaa gaaaaggggg gattgggggg tacagtgcag
     4861 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     4921 ttacaaaaat tcaaaatttt atcgatcacg agactagcct cgagctggct gcagtaacgc
     4981 cattttgcaa ggcatggaaa aataccaaac caagaataga gaagttcaga tcaagggcgg
     5041 gtacatgaaa atagctaacg ttgggccaaa caggatatct gcggtgagca gtttcggccc
     5101 cggcccgggg ccaagaacag atggtcaccg cagtttcggc cccggcccga ggccaagaac
     5161 agatggtccc cagatatggc ccaaccctca gcagtttctt aagacccatc agatgtttcc
     5221 aggctccccc aaggacctga aatgaccctg cgccttattt gaattaacca atcagcctgc
     5281 ttctcgcttc tgttcgcgcg cttctgcttc ccgagctcta taaaagagct cacaacccct
     5341 cactcggcgc gccagtcctc cgacagactg agtcggccgg tcgaatcaag cttatcgata
     5401 ccgtcgacgg atccatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca
     5461 aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
     5521 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc
     5581 ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc
     5641 accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc
     5701 gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg
     5761 acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg
     5821 taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg
     5881 gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg
     5941 tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg
     6001 tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     6061 agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaaggagg
     6121 tggcgcccga gcagccgcgc tggatggcgc acccggccgt gctgaatgcg cagcaccccg
     6181 actcacacca cccgggcctg gcgcacaact acatggaacc cgcgcagctg ctgcctccag
     6241 acgaggtgga cgtcttcttc aatcacctcg actcgcaggg caacccctac tatgccaacc
     6301 ccgctcacgc gcgggcgcgc gtctcctaca gccccgcgca cgcccgcctg accggaggcc
     6361 agatgtgccg cccacacttg ttgcacagcc cgggtttgcc ctggctggac gggggcaaag
     6421 cagccctctc tgccgctgcg gcccaccacc acaacccctg gaccgtgagc cccttctcca
     6481 agacgccact gcacccctca gctgctggag gccctggagg cccactctct gtgtacccag
     6541 gggctggggg tgggagcggg ggaggcagcg ggagctcagt ggcctccctc acccctacag
     6601 cagcccactc tggctcccac cttttcggct tcccacccac gccacccaaa gaagtgtctc
     6661 ctgaccctag caccacgggg gctgcgtctc cagcctcatc ttccgcgggg ggtagtgcag
     6721 cccgaggaga ggacaaggac ggcgtcaagt accaggtgtc actgacggag agcatgaaga
     6781 tggaaagtgg cagtcccctg cgcccaggcc tagctactat gggcacccag cctgctacac
     6841 accaccccat ccccacctac ccctcctatg tgccggcggc tgcccacgac tacagcagcg
     6901 gactcttcca ccccggaggc ttcctggggg gaccggcctc cagcttcacc cctaagcagc
     6961 gcagcaaggc tcgttcctgt tcagaaggcc gggagtgtgt caactgtggg gccacagcca
     7021 cccctctctg gcggcgggac ggcaccggcc actacctgtg caatgcctgt ggcctctacc
     7081 acaagatgaa tgggcagaac cgaccactca tcaagcccaa gcgaagactg tcggccgcca
     7141 gaagagccgg cacctgttgt gcaaattgtc agacgacaac caccacctta tggcgccgaa
     7201 acgccaacgg ggaccctgtc tgcaacgcct gtggcctcta ctacaagctg cacaatgtta
     7261 acaggccact gaccatgaag aaggaaggga tccagactcg gaactggaag atgtccaaca
     7321 agtccaagaa gagcaagaaa ggggcggagt gcttcgagga gctgtcaaag tgcatgcagg
     7381 agaagtcatc ccccttcagt gcagctgccc tggctggaca catggcacct gtgggccacc
     7441 tcccgccctt cagccactcc ggacacatcc tgcccactcc gacgcccatc cacccctcct
     7501 ccagcctctc cttcggccac ccccacccgt ccagcatggt gaccgccatg ggctagggat
     7561 cccccgggct gcaggaattc gagcatctta ccgccattta ttcccatatt tgttctgttt
     7621 ttcttgattt gggtatacat ttaaatgtta ataaaacaaa atggtggggc aatcatttac
     7681 atttttaggg atatgtaatt actagttcag gtgtattgcc acaagacaaa catgttaaga
     7741 aactttcccg ttatttacgc tctgttcctg ttaatcaacc tctggattac aaaatttgtg
     7801 aaagattgac tgatattctt aactatgttg ctccttttac gctgtgtgga tatgctgctt
     7861 taatgcctct gtatcatgct attgcttccc gtacggcttt cgttttctcc tccttgtata
     7921 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg
     7981 tgtgctctgt gtttgctgac gcaaccccca ctggctgggg cattgccacc acctgtcaac
     8041 tcctttctgg gactttcgct ttccccctcc cgatcgccac ggcagaactc atcgccgcct
     8101 gccttgcccg ctgctggaca ggggctaggt tgctgggcac tgataattcc gtggtgttgt
     8161 cggggaagct gacgtccttt gcaattcgat atcaagctgt acctttaaga ccaatgactt
     8221 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     8281 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     8341 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     8401 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     8461 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     8521 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     8581 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     8641 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     8701 tctggctcta gctatcccgc ccctaactc
//
