LOCUS       sequence-421753-        7898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for genetic tracing of mesenchymal Glioblastoma
            cell-state..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7898)
  AUTHORS   Company C, Schmitt MJ, Dramaretska Y, Serresi M, Kertalli S, Jiang 
            B, Yin JA, Aguzzi A, Barozzi I, Gargiulo G
  TITLE     Logical design of synthetic cis-regulatory DNA for genetic tracing 
            of cell identities and state changes.
  JOURNAL   Nat Commun. 2024 Feb 5;15(1):897. doi: 10.1038/s41467-024-45069-6.
  PUBMED    38316783
REFERENCE   2  (bases 1 to 7898)
  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: "2024-02-5"; volume: "15(1):897. doi"; pages: " 
            10.1038/s41467-024-45069-6"
COMMENT     Sequence Label: pPB-PNGT3-d2EGFP_CMV-mCherry
FEATURES             Location/Qualifiers
     source          1..7898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    316..336
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1398..1422
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1423..1432
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1429..1431,1432..1434,1435..2145,2146..2151,
                     2152..2274)
                     /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(1474..1495)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1735..1754)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2082..2103
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2275..2299)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2377..2396)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2442..2497
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2496..2515)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        2852..3231
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3232..3435
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3386..3406
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3462..3471
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3468..4178
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3613..3631)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3881..3900
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4216..4233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4222..4446
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4796..4858
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4924..4943)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4925..4943)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4950..4967)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4950..4966)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4959..4981)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5108..5563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5195..5214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5405..5426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5589..5693
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5694..5762,5763..6554)
                     /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(5912..5931)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6725..7313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7214..7233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7467..7484
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7601..7622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7637..7642,7643..7660,7661..7667)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7675..7691
                     /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     7680..7702
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7699..7715
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7699..7715
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7736..7754
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat atcatcaact ttgtatagaa aagttgtcca tctggtagcc taccctgagg
      361 ctttatttta tcaccctact tgtcccataa agattcttga gatttttatt gaaagaaaca
      421 atgaaaatgc taattagtta tatttttcat cataatcttt tttttatact aaaggatctc
      481 tctccttggt ctcaaactcc tgacctcagg tgatctgccc accttggcct cccaaagtgc
      541 taggattaca ggtgtgagcc actgcacctg gccctcccaa tatatttatg tgcttttttc
      601 tcccttattt tgttgctatg gtaatttatt agatattatg catttgattg actgtatttt
      661 attacctctc acttgttgaa acttagaaaa ctgactttca cggaagaagt cctagttgcc
      721 taatcaataa catgggtacc tctttgctcc ctgaaactac agagaatatt ctagagaatc
      781 agaaagtact tcccctaacc cccgctgttt tcacccccag cggcaacctt tgaatgcact
      841 tttgtttaca aacaccttcg ccagctattt tacgaccgtc tcgacaggaa gtgtgaatct
      901 gggttggaaa atccggtcaa tttgacccac ctacccaact tgcttctggt tcagaggtca
      961 cctgaagtta aggtgcttgt gtatacctta aggaattatg ccagatctag agttacatgt
     1021 tgattattaa taattaagaa caaagtgtca aaattatgta atcttttctc actttacaag
     1081 ctttgcataa agcattttta tcatgaatgg gtgttggatt ttgtcagctg ctttttctgc
     1141 atctattgat atgttcgaat gatatttttg tctttatact gttggtataa tgaattgcat
     1201 taattgatta tcaactagcc ttgtatatct ggaataaatc ccgggtcccg gggggcggcg
     1261 ctccacgcgg gttgtggggg gcgggggcgg cacgtgccgc cgctctcggc caatgcggag
     1321 ccccgcgcgg aggtcacgtg cctctgtttg gcgcttttgt gcgcgcccgg gtctgttggt
     1381 gctcagagtg tggttctaca agtttgtaca aaaaagcagg ctgccaccat ggtgagcaag
     1441 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1501 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1561 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1621 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1681 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1741 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1801 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1861 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1921 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1981 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2041 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2101 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagaagct tagccatggc
     2161 ttcccgccgg aggtggagga gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag
     2221 agcgggatgg accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt gtagacccag
     2281 ctttcttgta caaagtggtg atcctcaggt gcaggctgcc tatcagaagg tggtggctgg
     2341 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta
     2401 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     2461 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc
     2521 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     2581 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc
     2641 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     2701 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     2761 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag
     2821 atccctcgac ctgcagccca agcttcgcgt tgacattgat tattgactag ttattaatag
     2881 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     2941 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     3001 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     3061 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     3121 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     3181 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     3241 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     3301 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     3361 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     3421 tatataagca gagctctctg gctaactaga gaacccactg cgccaccatg gtgagcaagg
     3481 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     3541 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     3601 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     3661 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     3721 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     3781 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     3841 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     3901 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     3961 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     4021 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     4081 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     4141 gccactccac cggcggcatg gacgagctgt acaagtaact cgagtctaga gggcccgttt
     4201 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4261 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4321 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4381 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4441 ctatggctcg agttaattaa cgagagcata atattgatat gtgccaaagt tgtttctgac
     4501 tgactaataa gtataatttg tttctattat gtataggtta agctaattac ttattttata
     4561 atacaacatg actgttttta aagtacaaaa taagtttatt tttgtaaaag agagaatgtt
     4621 taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa
     4681 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     4741 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     4801 gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     4861 aaataatagt ttctaatttt tttattattc agcctgctgt cgtgaatacc gagctccaat
     4921 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
     4981 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5041 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5101 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5161 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5221 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5281 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5341 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5401 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5461 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5521 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5581 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5641 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5701 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5761 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5821 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5881 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5941 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6001 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6061 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6841 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7381 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7441 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7501 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7561 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     7621 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     7681 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     7741 accctcacta aagggaacaa aagctggtac ctcgcgcgac ttggtttgcc attctttagc
     7801 gcgcgtcgcg tcacacagct tggccacaat gtggtttttg tcaaacgaag attctatgac
     7861 gtgtttaaag tttaggtcga gtaaagcgca aatctttt
//
