LOCUS       sequence-477300-        7380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destabilized green fluorescent protein (d2EGFP) under 
            transcriptional control of NF-kB activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7380)
  AUTHORS   Campbell CA, Calderon R, Pavani G, Cheng X, Barakat R, Snella E, Liu
            F, Peng X, Essner JJ, Dorman KS, McGrail M, Gadue P, French DL, 
            Espin-Palazon R
  TITLE     p65 signaling dynamics drive the developmental progression of 
            hematopoietic stem and progenitor cells through cell cycle 
            regulation.
  JOURNAL   Nat Commun. 2024 Sep 6;15(1):7787. doi: 10.1038/s41467-024-51922-5.
  PUBMED    39242546
REFERENCE   2  (bases 1 to 7380)
  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: "2024-09-6"; volume: "15(1):7787. doi"; pages: " 
            10.1038/s41467-024-51922-5"
COMMENT     Sequence Label: 6xHsa.NFKB:d2EGFP
FEATURES             Location/Qualifiers
     source          1..7380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(32..43,44..206)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    585..609
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(747..771)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(937..948)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     regulatory      1009..1018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1015..1017,1018..1020,1021..1731,1732..1737,
                     1738..1860)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(1060..1081)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1321..1340)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1668..1689
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1870..2004
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1894..1913)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1948..1967
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(2443..2630,2631..2642)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2676..2692)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2692)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2729..2749)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2729..2747)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2768..2784)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2768..2784)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2781..2803)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2792..2808
                     /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(2816..2822,2823..2840,2841..2846))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2861..2882
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2999..3016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3170..3758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3250..3269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3929..4720,4721..4789))
                     /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     4552..4571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4790..4894)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4920..5375)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5057..5078)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5269..5288
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5502..5524
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5516..5533
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5517..5533
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5543..5562
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5543..5561
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 acaggtcaga ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt
       61 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct
      121 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat
      181 taaggaagta aaagtaaaag caagaaagaa aactagagat tcttgtttaa gcttttaatc
      241 tcaaaaaaca ttaaatgaaa tgcatacaag gttttatcct gctttagaac tgtttgtatt
      301 taattatcaa actataagac agacaatcta atgccagtac acgctactca aagttgtaaa
      361 acctcagatt taacttcagt agaagctgat tctcaaaatt gttagtgtca agcctagctc
      421 ttttggggct gaaaagcaat cctgcagtgc tgaaaagcct ctcacaggca gccgatgcgg
      481 gaagaggtgt attagtcttg atagagaggc tgcaaatagc aggaaacgtg agcagagact
      541 ccctggtgtc tgaaacacag gccagatggg ccctcgagat atcaacaagt ttgtacaaaa
      601 aagcaggctt cctgtgaatg catgcacggt cgagggactt tccgctgggg actttccgtc
      661 gagggacttt ccgctgggga ctttccgtcg agggactttc cgctggggac tttccgtcga
      721 cctgcagaat tcgaagcttg agctcgaccc agctttcttg tacaaagtgg ttgatatcca
      781 gtgacgtagg aagtccatcc attcacagcg cttctataaa ggcgccagct gaggcgccta
      841 ctactccaac cgcgactgca gcgagcaact gagaagactg gatagagccg gcggttccgc
      901 gaacgagcag tgaccgcgct cccacccagc tctgctctgc agctcccacc agtgtctacc
      961 cctggacccc ttgccgggct ttccccaaac ttcgaggatc caccggtcgc caccatggtg
     1021 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1081 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1141 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1201 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1261 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1321 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1381 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1441 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1501 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1561 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1621 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1681 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gaagcttagc
     1741 catggcttcc cgccggaggt ggaggagcag gatgatggca cgctgcccat gtcttgtgcc
     1801 caggagagcg ggatggaccg tcaccctgca gcctgtgctt ctgctaggat caatgtgtag
     1861 atcgatgatg atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     1921 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1981 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     2041 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     2101 gattatgatc ctctagatca gatctgcgaa gatacggcca cgggtgctct tgatcctgtg
     2161 gctgattttg gactgtgctg ctcgcagctg ctgatgaatc acatacttcc tccattttct
     2221 tccactgatt gactgttata atttccctaa tttccaggtc aaggtgctgt gcattgtggt
     2281 aatagatgtg acatgacgtc acttccaaag gaccaatgaa catgtctgac caatttcata
     2341 taatgtgaaa acgattttca taggcagaat aaataacatt taaattaaac tgggcatcag
     2401 cgcaattcaa ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat
     2461 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     2521 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     2581 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     2641 tggttcttga ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc
     2701 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     2761 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2821 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2881 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2941 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3001 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3061 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3121 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3181 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3241 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3301 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3361 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3421 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3481 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3541 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3601 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3661 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3721 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3781 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3841 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3901 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3961 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4021 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4081 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4141 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4201 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4261 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4321 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4381 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4441 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4501 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4561 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4621 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4681 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4741 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4801 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4861 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     4921 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     4981 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5041 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5101 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5161 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5221 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5281 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5341 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     5401 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     5461 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     5521 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccaaata
     5581 gtaggaatta cccacctgta caagtgctga aaacttggat gaataagccc gtttgccatt
     5641 ttctactgct attttaaatc ttttctgtat ttgtctgcat ttgtctttac ccttgaataa
     5701 atgttttagt tgtttttttt caattatgac tgtgtttaac agacattatt acaataatat
     5761 gtaatagtag tacacactat tattatgtaa tagtacttgt tgactgtatt tgagaactgg
     5821 accgagtgag tgttacgtca cccattcaaa atgacttact tctggctcca acaaaatgaa
     5881 gttaattcag ttgccatttt tcactgtatg gacatcgccg tgttggagct agacgttgcc
     5941 aataagcaag aaagagccga gatgcgtcga gtctgagtca ccgttcctat ggcaacccct
     6001 ctaaccaatc agaagtaagc ttgttggaag tccacagcct accacttgaa agcgggctgc
     6061 acaaaatctg tcaaacgttt tgaacgttgg atgtgagagc acatactttt attaaggcat
     6121 ctggttggtc agtttataat atcaacaact tgggctacag aaaagaaaag ttattacaga
     6181 aattatgatt aacaagtaca tgttaaataa agattttaat atgaatgcca ccactggagc
     6241 attcatgcca tttggagctt cttcctgttt ggatcactag aaggaggagg tcactcatta
     6301 cagttctcat atacagtcgt tggttggttg gttggttggt agattgattg attgattgat
     6361 tgattgattg attgattgat tgattgattg attgattgat tgattgattg attgattggt
     6421 agtcaaaata agaaataatt tccacagatt cattacagaa atgattaaat gcatacataa
     6481 aaaactgggg ggggggggga tacaacaaca cacttaagtc acatttgcct acgtaaagaa
     6541 aagtaaagaa aatcaatagc tatattttac atctcctttt tttgctgtct tttaattagc
     6601 cttgttttgc tgttatcttg attgaaactg taacttcttc acctgcttct tttctttgta
     6661 ggttttgcca gcccagaaac aggcatggct gtgcaaggcc agagcaccat gcacaatgcg
     6721 ctgcatgtct tcatgaacgg ctcaatgtcc tcagtccagg gctcagccaa cgaccccatc
     6781 ttcctccttc accatgcttt cattgacagg taacaaacac gtcatgacat tagactgcac
     6841 agtttttgac aaagttcata caatctgttg tttatagctg ctacaattag tgaagtttgt
     6901 gaatgtactt ggatgagcag cgaaagatca attgagatca attgttagag tttggttgcc
     6961 ctgcagagca aagaacaaaa aataatctgg tggctttact gcgtgaggtt attattggtg
     7021 gaatagaaac acaaaacata attgcattta tttgtttaat tttttatctt atcttaactt
     7081 tcatcttgca tatttgtttc ttacatcatt tctagcatct ttgagcgctg gctaagaact
     7141 catcagcctc cccggtccat ctacccacgt accaatgcac caattggcca caatgacggc
     7201 tactacatgg tgccattcct tcctctttat aggaatggag actacctcct gtccaacaat
     7261 gctcttggat acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg
     7321 ccctctgatc tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac
//
