LOCUS       sequence-382430-        8729 bp    DNA     circular SYN 15-APR-2025
DEFINITION  constitutive expression of mCherry fused to human Gata2 with 
            mutation R362Q 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-R362Q
FEATURES             Location/Qualifiers
     source          1..8729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(140..159)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(141..159)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(169..186)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(169..185)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(178..200)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      327..782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(414..433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     624..645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        808..912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(913..981,982..1773)
                     /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(1131..1150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1944..2532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2433..2452
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2686..2703
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2820..2841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2856..2861,2862..2879,2880..2886)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2894..2910
                     /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     2899..2921
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2918..2934
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2918..2934
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2953..2973
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2955..2973
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3001..3227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3228..3408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3455..3580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4073..4306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4491..4535
                     /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    4802..4919
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4953..5359
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             5394..6095
                     /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(5527..5545)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5795..5814
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5989..6012
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    7752..8155
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     LTR             8248..8481
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8553..8674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8590..8609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8644..8663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      join(8714..8729,1..120)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
       61 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
      121 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
      181 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      241 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      301 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
      361 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      421 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      481 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      541 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      601 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      661 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      721 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      781 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      841 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
      901 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
      961 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1021 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1081 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1141 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1201 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1261 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1321 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1381 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1441 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1501 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1561 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     1621 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     1681 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     1741 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     1801 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     1861 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1921 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1981 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2041 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2101 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2161 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2221 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2281 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2341 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2401 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2461 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2521 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2581 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2641 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2701 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2761 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     2821 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     2881 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     2941 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
     3001 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
     3061 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
     3121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
     3181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
     3241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     3301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     3361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     3421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     3481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     3541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     3601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     3661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     3721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     3781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     3841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     3901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     3961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     4021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     4081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     4141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     4201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     4261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     4321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     4381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     4441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     4501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     4561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     4621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     4681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     4741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggttaa
     4801 cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     4861 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta
     4921 tcgatcacga gactagcctc gagctggctg cagtaacgcc attttgcaag gcatggaaaa
     4981 ataccaaacc aagaatagag aagttcagat caagggcggg tacatgaaaa tagctaacgt
     5041 tgggccaaac aggatatctg cggtgagcag tttcggcccc ggcccggggc caagaacaga
     5101 tggtcaccgc agtttcggcc ccggcccgag gccaagaaca gatggtcccc agatatggcc
     5161 caaccctcag cagtttctta agacccatca gatgtttcca ggctccccca aggacctgaa
     5221 atgaccctgc gccttatttg aattaaccaa tcagcctgct tctcgcttct gttcgcgcgc
     5281 ttctgcttcc cgagctctat aaaagagctc acaacccctc actcggcgcg ccagtcctcc
     5341 gacagactga gtcggccggt cgaatcaagc ttatcgatac cgtcgacgga tccatggtga
     5401 gcaagggcga ggaggtcatc aaagagttca tgcgcttcaa ggtgcgcatg gagggctcca
     5461 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     5521 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     5581 ccccccagtt catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt
     5641 acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     5701 gcggtctggt gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg
     5761 tgaagatgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag aagaccatgg
     5821 gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc
     5881 accaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag accatctaca
     5941 tggccaagaa gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca
     6001 cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc
     6061 acctgttcct gtacggcatg gacgagctgt acaaggaggt ggcgcccgag cagccgcgct
     6121 ggatggcgca cccggccgtg ctgaatgcgc agcaccccga ctcacaccac ccgggcctgg
     6181 cgcacaacta catggaaccc gcgcagctgc tgcctccaga cgaggtggac gtcttcttca
     6241 atcacctcga ctcgcagggc aacccctact atgccaaccc cgctcacgcg cgggcgcgcg
     6301 tctcctacag ccccgcgcac gcccgcctga ccggaggcca gatgtgccgc ccacacttgt
     6361 tgcacagccc gggtttgccc tggctggacg ggggcaaagc agccctctct gccgctgcgg
     6421 cccaccacca caacccctgg accgtgagcc ccttctccaa gacgccactg cacccctcag
     6481 ctgctggagg ccctggaggc ccactctctg tgtacccagg ggctgggggt gggagcgggg
     6541 gaggcagcgg gagctcagtg gcctccctca cccctacagc agcccactct ggctcccacc
     6601 ttttcggctt cccacccacg ccacccaaag aagtgtctcc tgaccctagc accacggggg
     6661 ctgcgtctcc agcctcatct tccgcggggg gtagtgcagc ccgaggagag gacaaggacg
     6721 gcgtcaagta ccaggtgtca ctgacggaga gcatgaagat ggaaagtggc agtcccctgc
     6781 gcccaggcct agctactatg ggcacccagc ctgctacaca ccaccccatc cccacctacc
     6841 cctcctatgt gccggcggct gcccacgact acagcagcgg actcttccac cccggaggct
     6901 tcctgggggg accggcctcc agcttcaccc ctaagcagcg cagcaaggct cgttcctgtt
     6961 cagaaggccg ggagtgtgtc aactgtgggg ccacagccac ccctctctgg cggcgggacg
     7021 gcaccggcca ctacctgtgc aatgcctgtg gcctctacca caagatgaat gggcagaacc
     7081 gaccactcat caagcccaag cgaagactgt cggccgccag aagagccggc acctgttgtg
     7141 caaattgtca gacgacaacc accaccttat ggcgccaaaa cgccaacggg gaccctgtct
     7201 gcaacgcctg tggcctctac tacaagctgc acaatgttaa caggccactg accatgaaga
     7261 aggaagggat ccagactcgg aaccggaaga tgtccaacaa gtccaagaag agcaagaaag
     7321 gggcggagtg cttcgaggag ctgtcaaagt gcatgcagga gaagtcatcc cccttcagtg
     7381 cagctgccct ggctggacac atggcacctg tgggccacct cccgcccttc agccactccg
     7441 gacacatcct gcccactccg acgcccatcc acccctcctc cagcctctcc ttcggccacc
     7501 cccacccgtc cagcatggtg accgccatgg gctagggatc ccccgggctg caggaattcg
     7561 agcatcttac cgccatttat tcccatattt gttctgtttt tcttgatttg ggtatacatt
     7621 taaatgttaa taaaacaaaa tggtggggca atcatttaca tttttaggga tatgtaatta
     7681 ctagttcagg tgtattgcca caagacaaac atgttaagaa actttcccgt tatttacgct
     7741 ctgttcctgt taatcaacct ctggattaca aaatttgtga aagattgact gatattctta
     7801 actatgttgc tccttttacg ctgtgtggat atgctgcttt aatgcctctg tatcatgcta
     7861 ttgcttcccg tacggctttc gttttctcct ccttgtataa atcctggttg ctgtctcttt
     7921 atgaggagtt gtggcccgtt gtccgtcaac gtggcgtggt gtgctctgtg tttgctgacg
     7981 caacccccac tggctggggc attgccacca cctgtcaact cctttctggg actttcgctt
     8041 tccccctccc gatcgccacg gcagaactca tcgccgcctg ccttgcccgc tgctggacag
     8101 gggctaggtt gctgggcact gataattccg tggtgttgtc ggggaagctg acgtcctttg
     8161 caattcgata tcaagctgta cctttaagac caatgactta caaggcagct gtagatctta
     8221 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     8281 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     8341 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     8401 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     8461 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
     8521 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     8581 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     8641 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     8701 cctaactccg cccatcccgc ccctaactc
//
